EDG8 receptor, its preparation and use

ABSTRACT

The present invention relates to newly identified human EDG8 receptors, polynucleotides encoding this receptor, polypeptides encoded by such polynucleotides, the preparation and the use of such polynucleotides and polypeptides.

[0001] This application claims priority to European Patent ApplicationNos. 108858.2, filed Apr. 26, 2000, and 116589.3, filed Aug. 1, 2000,the disclosures of which are hereby incorporated by reference in theirentireties.

FIELD OF THE INVENTION

[0002] The present invention relates to newly identified human EDG8receptors, polynucleotides encoding this receptor, polypeptides encodedby such polynucleotides, the preparation and the use of suchpolynucleotides and polypeptides.

BACKGROUND OF THE INVENTION

[0003] In an effort to identify new G-protein coupled receptors of theEDG (endothelial differentiation gene)-family a novel member of theEDG-family of G-protein coupled receptors, Human EDG8, was identified.The full-length clone was isolated and studies on chromosomal mapping,tissue expression and identification as a functional cellular receptorfor sphingosine 1-phosphate were performed. Taken together, the dataprovide compelling evidence that EDG8 is the fifth receptor forsphingosine 1-phosphate, exclusively expressed in peripheral tissues,its presence in endothelial cells being responsible for the broad tissuedistribution.

[0004] The lysolipid phosphate mediators lysophosphatidic acid (LPA) andsphingosin 1-phosphate (S1P) have attracted increasing attention asmodulators of a variety of important biological functions (Moolenaar etal., Current Opinion in Cell Biol 9:168, 1997; Morris, Trends PharmacolSci 20:393, 1999; Lynch and Im, Trends in Pharmacol Sci 20:473,1999) andtheir list of biological activities is continuously growing.

[0005] Among the biological responses to LPA is platelet aggregation(Jalink et al., Biochem Biophys Acta 1198:185,1994; Siess et al., PNASUSA 96:6931, 1999; Gueguen et al., Biochemistry 38: 8440, 1999), smoothmuscle contraction (Tokumura et al., Arch Int Pharmacodyn Ther 245:74,1980), in vivo vasoactive effects (Tokumura et al., Res Comm Mol PatholPharmacol 90:96,1995), chemotaxis (Jalink et al., PNAS USA 90:1857,1993), expression of adhesion molecules (Lee et al., J Biol Chem273:22105, 1998; Rizza et al., Laboratory Investigation 79:1227, 1999),increased tight junction permeability of endothelial cells (Schulze etal., J Neurochem 68:991,1997), induction of stress fibers (Gohla et al.,J Biol Chem 274:17901, 1998) and many others (for review see Moolenaaret al., Current Opinion in Cell Biol 9:168, 1997). The biochemicalsignalling events that mediate the cellular effects of LPA includestimulation of phospholipases, mobilization of intracellular Ca²⁺,inhibition of adenylyl cyclase, activation of phosphatidylinositol3-kinase, activation of the Ras-Raf-MAP kinase cascade and stimulationof Rho-GTPases (Moolenaar et al., Current Opinion in Cell Biol 9:168,1997).

[0006] S1P, in particular, is implicated in cell proliferation,modulation of cell motility (reviewed in Hla et al., Biochem Pharm58:201, 1999) induction/suppression of apoptosis (Hisano et al., Blood93:4293, 1999; Xia et al., J Biol Chem 274:34499, 1999), angiogenesis(Lee et al., Cell 99:301, 1999), tumor invasiveness (Sadahira et al.,PNAS USA 89:9686, 1992), platelet activation (Gueguen et al.,Biochemistry 38: 8440,1999) and neurite retraction (Postma et al., EMBOJ 15:2388, 1996). Cellular signalling by S1P involves activation of PLCβand subsequent intracellular Ca²⁺ release (van Koppen et al., J BiolChem 271:2082, 1996; Meyer zu Heringdorf et al., Naunyn-Schmiedeberg'sArch Pharmacol 354:397,1997; Yatomi et al., J Biol Chem 272:5291, 1997a;Noh et al., J Cell Physiol 176:412,1998; Ancellin and Hla, J Biol Chem274:18997,1999), activation of MAP-kinases (Wu et al., J Biol Chem270:11484,1995; Lee et al., J Biol Chem 271:11272,1996; An et al., JBiol Chem 275:288, 2000), activation of inward rectifying K⁺-channels(van Koppen et al., J Biol Chem 271:2082, 1996; Bünemann et al., EMBO J15:5527,1996) and inhibition and/or activation of adenylyl cyclase (Leeet al., J Biol Chem 271:11272, 1996).

[0007] Both, LPA and S1P are recognized to signal cells through a set ofG-protein coupled receptors (GPCRs) known as EDG (endothelialdifferentiation gene)-receptors. The EDG-family of GPCRs currentlycomprises seven human members (EDG1-7) that fall into two major groupsdepending on their preference for the activating lipid-ligand: EDG1, 3,5 and 6 preferentially interact with S1P (Yatomi et al., J Biochem(Tokyo) 12:969, 1997; Lee et al., Science 279:1552, 1998; Lee et al., JBiol Chem 273:22105, 1998; Ancellin and Hla, J Biol Chem 274:18997,1999; Yamazaki et al., Biochem Biophys Res Commun 268:583, 2000; VanBrocklyn et al., Blood 95:2624, 2000), EDG2, 4 and 7 preferentiallyinteract with LPA (An et al., J Biol Chem 273:7906,1998; Im et al., MolPharmacol 57:753, 2000).

[0008] The assignment of specific biological functions to certainreceptor subtypes is hampered by the fact that EDG receptors areexpressed in an overlapping fashion (Rizza et al., LaboratoryInvestigation 79:1227, 1999; Lee et al., Cell 99:301, 1999), theyactivate multiple and in part redundant signal transduction pathways(Lee et al., J Biol Chem 271:11272, 1996; Ancellin and Hla, J Biol Chem274:18997, 1999; Kon et al., J Biol Chem 274:23940,1999; An et al., JBiol Chem 275:288, 2000), the selectivity for their activating ligandsis not absolute (Lee et al., J Biol Chem 273:22105, 1998), and medicinalchemistry is only poorly developed in that specific antagonists fordissecting the pharmacology of the individual subtypes are not availableyet.

[0009] An important step to shed more light on the biological role ofthe individual receptor subtypes would be to identify the complete setof receptors that respond to the phospholipid mediators S1P and LPA.

SUMMARY OF THE INVENTION

[0010] The present invention relates to newly identified human EDG8receptors, polynucleotides encoding this receptor, polypeptides encodedby such polynucleotides and the preparation and the use thereof.

[0011] The present invention relates to an isolated polynucleotidecomprising a nucleotide sequence that has at least 90% identity,preferably 95% or more, most preferably 98% identity to a nucleotidesequence encoding the polypeptide of SEQ ID NO:2 or the correspondingfragment thereof; or a nucleotide sequence complementary to saidnucleotide sequence.

[0012] The present invention also relates to an isolated polynucleotidecomprising a nucleotide sequence that has at least about 90% identity,preferably about 95% or more, most preferably about 98% identity to anucleotide sequence encoding the polypeptide of SEQ ID NO:2 or thecorresponding fragment thereof; or a nucleotide sequence complementaryto said nucleotide sequence.

[0013] Preferably, the polynucleotide is DNA or RNA. The nucleotidesequence of the polynucleotide is at least 90% or about 90% identical tothat contained in SEQ ID NO:1; preferably 95% or about 95% or more, mostpreferred 98% or about 98% or more identical to SEQ ID NO:1. In anotherembodiment, the polynucleotide has the nucleotide sequence SEQ ID NO:1.In another embodiment, the polynucleotide encodes the polypeptide of SEQID NO:2 or a fragment thereof. In a special embodiment, thepolynucleotide is an allele of SEQ ID NO:1. Preferably, thepolynucleotide has the same essential properties and/or biologicalfunctionality as human EDG8.

[0014] One characteristic of “functionality” or “biologicalfunctionality” is that the polynucleotide encodes for a S1P receptor; itresponds to S1P and optionally also to related phospholipids like DMS 1Por LPA. By “functionality” is meant the molecule is a functionalreceptor for S1P, LPA, dHS1P and related lysophospholipid mediators.Such activity may be assayed using well known techniques in the art. Onesuch assay employs assessment of ability of Ca²⁺ mobilization inresponse to S1P mediated by the receptor, e.g., EDG8 or a functionalfragment thereof, in CHO cell as set forth in the description of FIG. 2.

[0015] Another aspect of the invention relates to an expression systemfor the expression of EDG8. The EDG8 DNA or RNA molecule comprising anexpression system wherein said expression system is capable of producinga polypeptide or a fragment thereof having at least 90% or about 90%identity, preferably 95% or about 95% or more, most preferred 98% orabout 98% or more identity with a nucleotide sequence encoding thepolypeptide of SEQ ID NO. 2 or said fragment when said expression systemis present in a compatible host cell. Preferably, the expression systemis a vector.

[0016] The invention relates to a host cell comprising the expressionsystem.

[0017] In another aspect, the invention relates to a process forproducing a human EDG8 polypeptide or a fragment thereof wherein a hostcell comprising the expression system is cultured under conditionssufficient for the production of said polypeptide or fragment thereof.Preferably, the said polypeptide or fragment thereof is expressed at thesurface of said cell.

[0018] The invention relates also to cells produced by this process.

[0019] The process preferably further includes recovering thepolypeptide or fragment thereof from the culture.

[0020] In another aspect, the invention relates to a process forproducing a cell which produces an EDG8 polypeptide or a fragmentthereof comprising transforming or transfecting a host cell with theexpression system such that the host cell, under appropriate cultureconditions, produces a human EDG8 polypeptide or a fragment thereof.

[0021] Thus, in one embodiment, the invention relates to an isolatedpolynucleotide comprising a polynucleotide selected from the groupconsisting of:

[0022] (a) a polynucleotide encoding the polypeptide consisting of theamino acid sequence of SEQ ID NO:2;

[0023] (b) a polynucleotide consisting of SEQ ID NO:1;

[0024] (c) a polynucleotide having at least about 90% sequence identityto the polynucleotide of (a) or (b).

[0025] In another embodiment, the invention relates to a fragment of thepolynucleotide of SEQ ID NO:1. In yet another embodiment, the inventionrelates to a polynucleotide which is a complement of the above describedpolynucleotide.

[0026] Other embodiments relate to an expression vector comprising theisolated polynucleotide and a host cell comprising such expressionvector. A further embodiment is a method of producing a polypeptidecomprising SEQ ID NO:2 by culturing such host cell under conditionssufficient for the production of the polypeptide and recovering it fromthe culture. Another embodiment of the invention relates to a processfor producing cells capable of expressing the above polypeptidecomprising genetically transfecting or transforming cells with the abovevector.

[0027] Another embodiment relates to an antibody that selectively bindsa polypeptide comprising the amino acid sequence of SEQ ID NO:2 or afragment of SEQ ID NO:2.

[0028] A further embodiment relates to a process for diagnosing adisease or a susceptibility to a disease related to expression oractivity of human EDG8 polypeptide comprising:

[0029] determining the presence or absence of mutation in the nucleotidesequence encoding human EDG8 polypeptide in the genome of the subject;and/or

[0030] analyzing for the presence or amount of the human EDG8polypeptide expression in a sample derived from the subject.

[0031] Another embodiment relates to a method for identifying compoundswhich bind to human EDG8 polypeptide comprising:

[0032] a) contacting a cell containing the above describedpolynucleotides of the invention with a candidate compound; and

[0033] b) assessing the ability of said candidate compound to bind tothe cells. This method further includes determining whether thecandidate compound effects a signal generated by activation of the humanEDG8 polypeptide at the surface of the cell, wherein a candidatecompound which effects production of the signal is identified as anagonist. In another embodiment, this method further includes determiningwhether the candidate compound effects a signal generated by activationof the human EDG8 polypeptide at the surface of the cell, wherein acandidate compound which effects production of the signal is identifiedas an antagonist.

[0034] Thus, the present invention relates to agonists and antagonistsidentified by the above described methods.

[0035] In yet another embodiment, the invention relates to a method ofpreparing a pharmaceutical composition comprising:

[0036] a) identifying a compound which is an agonist or an antagonist ofhuman EDG8,

[0037] b) preparing the compound, and

[0038] c) optionally mixing the compound with suitable additives and topharmaceutical composition prepared by such method.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039]FIG. 1A: The nucleotide and deduced amino acid sequence of humanEDG8. The deduced amino acid sequence is shown below the nucleotidesequence with the nucleotide positions indicated on the left.

[0040]FIG. 1B: Phylogenetic tree of the EDG-family of receptors. Thephylogenetic tree depicted was derived by the neighbor joining methodperformed with the GCG program Wisconsin package version 10.1-Unix(Genetic Computer Group (GCG), Madison, Wis.

[0041]FIG. 1C: Alignment of the amino acid sequence of human EDG8 withthe other EDG-family members. The amino acid sequence of human EDG8(accession number AC011461) is compared with the EDG1-7 polypeptides(EDG1: accession number M 31210, EDG2: accession number U 80811, EDG3:accession number X 83864, EDG4: accession number AF 011466, EDG5:accession number AF 034780, EDG6: AJ 000479, EDG7: accession number AF127138). The approximate boundaries of the seven putative transmembranedomains are boxed. Gaps are introduced to optimize the alignment.

[0042] FIGS. 2A-F: Mobilization of intracellular Ca²⁺ by SIP (10, 100and 1000 nM) mediated by the EDG1, 3, 5, 6 and 8 receptor in CHO cells,cotransfected with empty vector DNA as a control or the indicatedG-protein a subunits.

[0043] A: S1P-induced Ca²⁺-response in CHO cells transfected with vectorDNA alone or the G protein a subunits Gq, G16 and Gqi5. B-F: S1P-inducedCa²⁺-response in CHO cells transfected with the indicated EDG-receptorsubtypes. Agonist-mediated changes of intracellular Ca²⁺ were measuredwith the FLIPR using the Ca²⁺-sensitive dye FLUO4 as described inExperimental procedures. Fluorescence of transfected cells loaded withFLUO4 was recorded before and after addition of S1P, applied in theindicated concentrations. Data are expressed as means of quadruplicatedeterminations in a single experiment. An additional experiment gavesimilar results.

[0044]FIG. 3: Effects of S1P, LPA and related lysophospholipid mediatorson EDG8-mediated increase in intracellular Ca²⁺. CHO-cells werecotransfected with EDG8 and the G protein a subunits Gqi5 (upper panel)and G16 (lower panel) and rises in [Ca²⁺]_(i) were recorded with theFLIPR as described in Experimental procedures. The different lipids wereapplied in concentrations of 10, 100 and 1000 nM, respectively. Data aremeans of quadruplicate determinations of a representative experiment.Two additional experiments gave similar results.

[0045]FIG. 4: Northern blot analysis of EDG8 in human tissues.Poly(A)+RNA (1 μg) from various human tissues (human multiple tissueNorthern blots, CLONTECH) was hybridized with probes specific to humanEDG8 (upper panel) and β-actin (lower panel) on a nylon membrane. Theorigin of each RNA is indicated at the top, the molecular mass ofstandard markers in kilobases (kb) is shown on the left.

[0046]FIG. 5A: Reverse transcriptase-polymerase chain reaction (RT-PCR)analysis of EDG8 in different human endothelial cell lines (HUVECs:human umbilical vein endothelial cells; HCAEC: human coronary arteryendothelial cells; HMVEC-L: human microvascular endothelial cells fromlung; HPAEC: human pulmonary artery endothelial cells). EDG8-specifictranscripts were detected in all endothelial cell lines. Agarose gelelectrophoresis of the PCR products after 35 cycles of amplificationwith the GC-melt kit (as described in Experimental Procedures) is shown.Amplification with EDG8-specific primers yields a 522 bp EDG8-fragmentas indicated by the arrow. The EDG8 plasmid served as a template for thepositive control, H₂O was used instead of plasmid DNA as a negativecontrol.

[0047]FIG. 5B: PCR analysis of EDG8 primers for specificity ofamplification of EDG8 sequences. Primers, specific for the EDG8sequence, were checked for potential amplification of the related EDG1-7sequences, using the respective plasmids as templates. Agarose gelelectrophoresis of the PCR products after 35 cycles of amplificationwith the GC-melt kit (as described in Experimental Procedures) is shown.The EDG8 specific 522 bp band occurred only when EDG8 was used as atemplate. H₂O was used instead of plasmid DNA as a negative control.

[0048]FIG. 6: Experiments were performed according to example 3. Insteadof lipids, a lipid library was used.

[0049] FIGS. 6A+B: Library plattes with rat EDG8 (r EDG8) and qi5.

[0050]FIG. 6A: qi5 background.

[0051]FIG. 6B: Measurement with rEDG8.

[0052]FIG. 6C: Fluorescence change counts.

[0053]FIG. 7: Experiments were performed according to example 3. Insteadof Lipids, a lipid library was used.

[0054] FIGS. 7A+B: Library plates with human EDG8 (hEDG8) and qi5.

[0055]FIG. 7A: q15 background.

[0056]FIG. 7B: Measurement with hEDG8

[0057]FIG. 7C: Fluourescence change counts.

[0058]FIG. 8: Antagonism of S1P activation of rat and human EDG8.Transiently transfected CHO cells expressing rat EDG8 and Gα_(qi5) (A)and HEK 293 cells expressing human EDG8 and Gα_(qi5) (B) were incubatedwith test compounds, namely, 0.1 μM Leukotriene B4, 1 μM 2-DHLA-PAF(1-O-Hexadecyl-2-O-dihomo-γ-linolenoyl-sn-glycero-3-phophorylcholine), 1μM C₂ Dihydroceramide, 0.1 μM 15(S) HEDE (15(S)-Hydroxyeicosa-11Z,13E-dienoic acid), 1 μM PAF C16(1-O-Hexadecyl-2-O-acetyl-sn-glycero-3-phosphorylcholine), 1 μM 16,16Dimethyl PGE₂ (16,16-Dimethyl-Prostaglandin E₂) 12, 0.1 μM (R)-HETE(12(R)-Hydroxyeicosa-5Z,8Z,10E,14Z-tetraenioc acid), 1 μM 8-epi-PGF_(2α)(8epi-Prostaglandin F_(2α)) 0.1 μM Leukotoxin A ((±) 9,10-EODE) or withsolvent buffer for 3 min and then challenged with 1 μM S1P (sphingosine1-phosphate). Peak fluorescence counts of cells preincubated withsolvent buffer and then stimulated with 1 μM S1P were set 100%.Fluorescence change counts were recorded with the FLIPR as described indetail in Experimetal procedures. Data are means ±SE of 2-3 independentexperiments.

[0059]FIG. 9: Inhibition of SIP mediated intracellular calcium releaseby suramin and NF023(8,8′-(carbonylbis(imino-3,1-phenylene))bis-(1,3,5-naphatlenetrisulfonicacid)) in cells transiently cotransfected with with human EDG8 andGα_(qi5) (A) and rat EDG8 and Gα_(qi5) (B). Transfected cells were firsttreated with the indicated concentrations of the inhibitor or solventbuffer for 3 minutes (NF023 and suramin did not show any effect on[Ca²⁺]_(i) mobilization during the preincubation period). Cells werethen stimulated with 1 μM SIP and in [Ca²⁺]_(i) measured with the FLIPRas described in the method section. Peak fluorescence counts werenormalized and background responses of Gα_(qi5)-transfected cells weresubtracted. SIP-mediated calcium release in the absence of inhibitor wasset 100%. Data are means ±SE of 4-7 independent experiments.

DETAILED DESCRIPTION OF THE INVENTION

[0060] The abbreviations used are:

[0061] SIP, sphingosine 1-phosphate; LPA, lysophosphatidic acid; dHS1P,dihydro sphingosine 1-phosphate; SPC, sphingosylphosphorylcholine; LPC,lysophosphatidylcholine; GPCR, G-protein-coupled receptor; G-protein,guanine nucleotide-binding protein; [Ca²⁺]_(i), intracellular Calciumconcentration, RT-PCR, reverse transcription polymerase chain reaction;bp, base pair; ORF, open reading frame; EST, expressed sequence tag;FAF-BSA, fatty acid free bovine serum albumine; HUVECs, Human umbilicalvein endothelial cells; HCAECs, human coronary artery endothelial cells;HMVEC-L, human microvascular endothelial cells from lung; HPAEC, humanpulmonary artery endothelial cells.

[0062] In particular, the invention relates to an EDG8 polypeptide or afragment thereof comprising an amino acid sequence which has at leastabout 90%, preferably at least about 95%, most preferred about 98% ormore identity to the amino acid sequence SEQ ID NO. 2 or to a part ofSEQ ID NO. 2. In particular the invention relates to an EDG8 polypeptideor a fragment thereof having amino acid sequence SEQ ID NO. 2 or a partthereof. In particular, the invention relates to an polypeptide encodedby SEQ ID NO. 1 or encoded by a polynucleotide that has at least about90%, preferably at least about 95%, most preferred about 98% or moreidentity with SEQ ID NO. 1; preferably, such polypeptide has almost thesame properties as human EDG 8; e.g. the same biological activity orfunctionality. One characteristic functionality of human EDG8 is thatthe polypeptide is a S1P receptor; it responds to S1P and optionally torelated phospholipids like dHS1P or LPA as depicted and described inFIG. 2.

[0063] In an additional embodiment, a method of detecting a nucleic acidsequence encoding SEQ ID NO:1 in a biological sample comprisingcontacting a labeled nucleic acid probe that hybridizes with the nucleicacid sequence with the biological sample under conditions wherein theprobe hybridizes with the nucleic acid sequence and detecting thehybridization of the probe to the nucleic acid sequence in the sample isprovided.

[0064] By “biological sample” is meant any body fluid, tissue, cells orspecimens obtained from a subject, such as from blood, urine, saliva,tissue biopsy and autopsy material. The genomic DNA from the biologicalsamples may be used directly or may be amplified enzymatically by usingPCR (Saiki et al., Nature 324:163-166, 1986) prior to analysis. RNA orcDNA may also be used for the same purpose. As an example, PCR primerscomplementary to the nucleic acid encoding the G-protein coupledreceptor protein can be used as a probe to identify and analyzeG-protein coupled receptors. The probe is labeled according to methodswell-known to the skilled artisan, which are described below. Similarly,the detecting is conducted under hybridization conditions that are knownto the skilled artisan and are describe further, below.

[0065] In an additional embodiment, the invention relates to a kitcomprising one or more containers, wherein at least one containercontains a detectably labeled antibody that selectively binds apolypeptide encoded by SEQ ID NO:1. A kit comprising one or morecontainers, wherein at least one container contains a detectably labelednucleic acid probe that hybridizes under a stringency of 68° C. with apolynucleotide encoding SEQ ID NO:2 is also provided.

[0066] The invention further relates to a polypeptide consisting of theamino acid sequence of SEQ ID NO. 2.

[0067] This invention is further related to a DNA sequence wherein theDNA sequence has been selected from at least one of the following groupof polynucleotide sequences:

[0068] a) a polynucleotide comprising the polynucleotide sequence of SEQID No 1,

[0069] b) a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide sequence according to a),

[0070] c) a polynucleotide which hybridizes under low or mediumstringency conditions to a polynucleotide sequence according to a), and

[0071] d) a polynucleotide sequence complementary to a polynucleotidesequence as defined in one of a), b), or c).

[0072] Thus, in one embodiment, the invention relates to an isolatednucleic acid molecule encoding a EDG8 polypeptide comprising SEQ IDNO:2. In another embodiment, the invention relates to an isolatednucleic acid molecule comprising SEQ ID NO:1, a nucleic acid moleculethat is at least 95% or about 95% dentical to SEQ ID NO:1, a nucleicacid molecule that hybridizes under stringent conditions to one of theabove or is complementary to one of the above. In another embodiment,the nucleic acid sequence consists of SEQ ID NO. 1.

[0073] The nucleic acid molecule of the present invention may be in theform of RNA, such as mRNA, or in the form of DNA, including, forinstance cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded. Singlestranded DNA or RNA may be the coding strand, also known as the sensestrand, or it may be the non-coding strand, also referred to as theanti-sense strand.

[0074] By “isolated” nucleic acid molecule is intended a nucleic acidmolecule, DNA or RNA, that has been removed from its native environment.For example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the DNA molecules of the presentinvention. Isolated nucleic acid molecules further includes suchmolecules produced synthetically.

[0075] In another embodiment, the DNA sequence as mentioned above can bepart of the genome of each organism which harbors a gene for EDG8. Inparticular, the DNA sequence is part of a mammal or a human being.

[0076] It is understood that all nucleic acid molecules encoding EDG8are also included herein, as long as they encode a polypeptide havingthe biological activity of human EDG8. By “EDG8 biological activity” ismeant that the molecule is a functional receptor for S1P, LPA, dHS1P andrelated lysophospholipid mediators. Such activity may be assayed usingwell known techniques in the art. One such assay employs assessment ofthe ability of Ca²⁺ to mobilize as described in FIG. 2. Such nucleicacid molecules include naturally occurring, synthetic, and intentionallymanipulated polynucleotides. For example, DNA encoding EDG8 may besubjected to site-directed mutagenesis. The nucleotide sequence for EDG8also includes antisense sequences, and sequences encoding dominantnegative forms of EDG8. The invention includes nucleotide sequences thatare degenerate as a result of the genetic code. There are 20 naturalamino acids, most of which are specified by more than one codon.Therefore, all degenerate nucleotide sequences are included in theinvention as long as the amino acid sequence of EDG8 polypeptide encodedby the nucleotide sequence is functionally unchanged. When the sequenceis RNA, the deoxynucleotides A, G, C, and T of SEQ ID NO:1 are replacedby ribonucleotides A, G, C, and U, respectively.

[0077] The present invention also includes fragments of the abovedescribed nucleic acid molecule. For instance, fragments include asegment of contiguous nucleotides of SEQ ID NO:1, which are at leastabout 10 bases, preferably about 15 bases or about 20 bases or 30 bases,or 40 bases, or 50 bases in length. Such fragments are useful asdiagnostic probes and PCR primers, as set forth herein. Of course,larger fragments of the nucleic acid molecules of the present inventionalso are contemplated. Fragments or portions of the polynucleotides ofthe present invention also may be used to synthesize full-lengthpolynucleotides of the present invention.

[0078] For example, a nucleic acid probe may be used to identify a cDNAclone corresponding to a full length transcript and a genomic clone orclones that contain the complete gene of the present invention includingregulatory and promoter regions, exons and introns. An example of ascreen of this type comprises isolating the coding region of the gene byusing the known DNA sequence to synthesize an oligonucleotide probe.Labeled oligonucleotides having a sequence complementary to that of thegenes of the present invention are used to screen a library of humancDNA, genomic DNA or mRNA to determine which members of the library theprobe hybridizes to.

[0079] The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

[0080] As described above, fragments of the full length gene of thepresent invention may be used as hybridization probes for a cDNA or agenomic library to isolate the full length DNA and to isolate other DNAswhich have a high sequence similarity to the gene or similar biologicalactivity. Probes of this type preferably have at least 10, preferably atleast 15, and even more preferably at least 30 bases and may contain,for example, at least 50 or more bases. In fact, probes of this typehaving at least up to 150 bases or greater may be preferably utilized.The probe may also be used to identify a DNA clone corresponding to afull length transcript and a genomic clone or clones that contain thecomplete gene including regulatory and promotor regions, exons andintrons. An example of a screen comprises isolating the coding region ofthe gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary or identical to that of the gene or portion of the genesequences of the present invention are used to screen a library ofgenomic DNA to determine which members of the library the probehybridizes to.

[0081] It is also appreciated that such probes can be and are preferablylabeled with an analytically detectable reagent to facilitateidentification of the probe. Useful reagents include but are not limitedto radioactivity, fluorescent dyes or enzymes capable of catalyzing theformation of a detectable product. The probes are thus useful to isolatecomplementary copies of DNA from other sources or to screen such sourcesfor related sequences.

[0082] Thus, the present invention is directed to polynucleotides havingat least about a about 70% identity, preferably at least about 90%identity and more preferably at least about a 95% identity to apolynucleotide which encodes the polypeptide of SEQ ID NO:2, as well asfragments thereof, which fragments have at least 15 bases, preferably atleast 30 bases, more preferably at least 50 bases and most preferablyfragments having up to at least 150 bases or greater, which fragmentsare at least about 90% identical, preferably at least about 95%identical and most preferably at least about 97% identical to anyportion of a polynucleotide of the present invention.

[0083] In another embodiment, the invention relates to a nucleic acidmolecule that hybridizes under stringent condition to SEQ ID NO:1. Thephrase “stringent hybridization conditions” refers to conditions underwhich a probe will hybridize to its target complementary sequence,typically in a complex mixture of nucleic acids, but to no othersequences. Stringent conditions are sequence-dependent andcircumstance-dependent; for example, longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993).

[0084] Generally, stringent conditions are selected to be about 5-10° C.lower than the thermal melting point (Tm) for the specific sequence at adefined ionic strength pH. The Tm is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at TR, 50%of the probes are occupied at equilibrium). Stringent conditions will bethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes (e.g., 10 to 50 nucleotides) and at least about 60° C. forlong probes (e.g., greater than 50 nucleotides). Stringent conditionsmay also be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, preferably 10 times backgroundhybridization.

[0085] Exemplary, non-limiting stringent hybridization conditions can beas following: 50% formamide, 5× SSC, and 1% SDS, incubating at 42° C.,or, 5× SSC, 1 SDS, incubating at 65° C., with wash in 0.2× SSC, and 0.1%SDS at 65° C. Alternative conditions include, for example, conditions atleast as stringent as hybridization at 68° C. for 20 hours, followed bywashing in 2× SSC, 0.1% SDS, twice for 30 minutes at 55° C. and threetimes for 15 minutes at 60° C. Another alternative set of conditions ishybridization in 6× at about 45° C., followed by one or more washes in0.2× SSC, 0.1% SDS at 50-65° C. For PCR, a temperature of about 36° C.is typical for low stringency amplification, although annealingtemperatures may vary between about 32° C. and 48° C. depending onprimer length. For high stringency PCR amplification, a temperature ofabout 62° C. is typical, although high stringency annealing temperaturescan range from about 50° C. to about 65° C., depending on the primerlength and specificity. Typical cycle conditions for both high and lowstringency amplifications include a denaturation phase of 90° C.-95° C.for 30 sec-2 min., an annealing phase lasting 30 sec.-2 min., and anextension phase of about 72° C. for 1-2 min.

[0086] Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1× SSC at 45° C. A positive hybridization is at leasttwice background. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

[0087] In an embodiment of the present invention, by “stringent”conditions is meant washing of filters in 0.1× SSC, 0.1% SDS; 2 timesfor about 30 min. at about 68° C. (or about 5° C. below meltingtemperature). “Low medium” hybridization conditions means: washing offilters in 2× SSC, 0.1% SDS; 2 times for about 30 min. at about 68° C.(or about 10° C. below melting temperature).

[0088] The polynucleotides which hybridize to the above describedpolynucleotides in a preferred embodiment encode polypeptides whichretain substantially the same biological function or activity as themature polypeptide encoded by the cDNAs of SEQ ID NO:1. For example,such polypeptide could function as a receptor for S1P and relatedcompounds, viz., LPA, dHS1P and related lysophospholipid mediators. Suchactivity may be assayed using well known techniques in the art. One suchassay employs assessment of ability of Ca²⁺ mobilization in response toS1P mediated by the receptor, e.g., EDG8 or a functional fragmentthereof, in CHO cell as described in FIG. 2.

[0089] The nucleic acid molecule of the invention includes the DNAencoding SEQ ID NO:2 and conservative variations of SEQ ID NO:2. Theterm “conservative variation” as used herein denotes the replacement ofan amino acid residue by another, biologically similar residue. Examplesof conservative variations include the substitution of one hydrophobicresidue such as isoleucine, valine, leucine or methionine for another,or the substitution of one polar residue for another, such as thesubstitution of arginine for lysine, glutamic for aspartic acid, orglutamine for asparagine, and the like. The term “conservativevariation” also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid provided that antibodies raised tothe substituted polypeptide also immunoreact with the unsubstitutedpolypeptide.

[0090] The nucleic acid molecule of the present invention can be derivedfrom any mammal, particlarly humans. The preferred nucleic acid moleculeis derived from humans. In the present invention, the nucleic acidmolecule may be at least 95% or about 95% identical to SEQ ID NO:1. Oneof skill in the art can determine the percentage of sequence identitybetween two sequences by aligning the encoded amino acid sequences,determining the corresponding alignment of the encoding polynucleotides,and then counting the number of residues shared between the sequencesbeing compared at each aligned position. No penalty is imposed for thepresence of insertions or deletions, but insertion or deletions arepermitted only where required to accommodate an obviously increasednumber of amino acid residues in one of the sequences being aligned.Offsetting insertions just to improve sequence alignment are notpermitted at either the polypeptide or polynucleotide level. Thus, anyinsertions in the polynucleotide sequence will have a length which is amultiple of 3. The percentage is given in terms of residues in the testsequence that are identical to residues in the comparison referencesequence.

[0091] Percent identity is calculated for oligonucleotides of thislength by not allowing gaps in either the oligonucleotide or thepolypeptide for purposes of alignment. Whenever at least one of twosequences being compared is a degenerate oligonucleotide comprising anambiguous residue, the two sequences are identical if at least one ofthe alternative forms of the degenerate oligonucleotide is identical tothe sequence with which it is being compared. As an illustration, AYAAAis 100% identical to ATAAA, since AYAAA is a mixture of ATAAA and ACAAA.Methods to determine the homology and percent identity of sequences arewell known in the art. These methods can be performed manually (usingmathematical calculations) or with a computer program, such as theWisconsin package version 10.1-Unix (Genetics Computer Group (GCG),Madison, Wis.). Other methods of alignment of sequences for comparisonare well-known in the art. Optimal alignment of sequences for comparisonmay be conducted by the local homology algorithm of Smith and Waterman,Adv. Appl. Math. 2: 482 (1981); by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48: 443 (1970); by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. 8: 2444(1988); by computerized implementations of these algorithms, including,but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics,Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in theWisconsin Genetics Software Package, Genetics Computer Group (GCG), 7Science Dr., Madison, Wis., USA; the CLUSTAL program is well describedby Higgins and Sharp, Gene 73: 237-244 (1988); Higgins and Sharp,CABIOS: 11-13 (1989); Corpet, et al., Nucleic Acids Research 16: 881-90(1988); Huang, et al., Computer Applications in the Biosciences 8: 1-6(1992), and Pearson, et al., Methods in Molecular Biology 24: 7-331(1994). The BLAST family of programs which can be used for databasesimilarity searches includes: BLASTN for nucleotide query sequencesagainst nucleotide database sequences; BLASTX for nucleotide querysequences against protein database sequences; BLASTP for protein querysequences against protein database sequences; TBL ASTN for protein querysequences against nucleotide database sequences; and TBLASTX fornucleotide query sequences against nucleotide database sequences. See,Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York (1995).

[0092] Antibodies

[0093] In another embodiment of the invention, the EDG8 polypeptides ofthe invention, including fragments thereof, can be used to produceantibodies which are immunoreactive or bind to epitopes of the EDG8polypeptides. Polyclonal antibodies and antibodies which consistessentially of pooled monoclonal antibodies with different epitopicspecificities, as well as distinct monoclonal antibody preparations areencompassed by the invention.

[0094] The preparation of polyclonal antibodies is well-known to thoseskilled in the art. See, for example, Green et at., “Production ofPolyclonal Antisera,” in: Immunochemical Protocols pages 1-5, Manson,ed., Humana Press 1992; Coligan et al., “Production of PolyclonalAntisera in Rabbits, Rats, Mice and Hamsters,” in: Current Protocols inImmunology, section 2.4.1, 1992, which are hereby incorporated byreference.

[0095] The preparation of monoclonal antibodies likewise isconventional. See, for example, Kohler & Milstein, Nature 256:495, 1975;Coligan et al., sections 2.5.1-2.6.7; and Harlow et al., in: Antibodies:a Laboratory Manual, page 726, Cold Spring Harbor Pub., 1988, which arehereby incorporated by reference. Briefly, monoclonal antibodies can beobtained by injecting mice with a composition comprising an antigen,verifying the presence of antibody production by removing a serumsample, removing the spleen to obtain B lymphocytes, fusing the Blymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones that produce antibodies to theantigen, and isolating the antibodies from the hybridoma cultures.Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See,e.g., Coligan et al., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3;Barnes et al., “Purification of Immunoglobulin G (IgG),” in: Methods inMolecular Biology, Vol. 10, pages 79-104, Humana Press, 1992.

[0096] Methods of in vitro and in vivo multiplication of monoclonalantibodies are well known to those skilled in the art. Multiplication invitro may be carried out in suitable culture media is such as Dulbecco'sModified Eagle Medium or RPMI 1640 medium, optionally supplemented by amammalian serum such as fetal calf serum or trace elements andgrowth-sustaining supplements such as normal mouse peritoneal exudatecells, spleen cells, thymocytes or bone marrow macrophages. Productionin vitro provides relatively pure antibody preparations and allowsscale-up to yield large amounts of the desired antibodies. Large scalehybridoma cultivation can be carried out by homogenous suspensionculture in an airlift reactor, in a continuous stirrer reactor, or inimmobilized or entrapped cell culture. Multiplication in vivo may becarried out by injecting cell clones into mammals histocompatible withthe parent cells, e.g., syngeneic mice, to cause growth ofantibody-producing tumors. Optionally, the animals are primed with ahydrocarbon, especially oils such as pristane (tetramethylpentadecane)prior to injection. After one to three weeks, the desired monoclonalantibody is recovered from the body fluid of the animal.

[0097] Therapeutic applications for antibodies disclosed herein are alsopart of the present invention. For example, antibodies of the presentinvention may also be derived from subhuman primate antibody. Generaltechniques for raising therapeutically useful antibodies in baboons canbe found, for example, in Goldenberg et al., International PatentPublication WO 91/11465, 1991, and Losman et al., Int. J. Cancer 46:310,1990, which are hereby incorporated by reference.

[0098] Alternatively, a therapeutically useful anti- EDG8 antibody maybe derived from a “humanized” monoclonal antibody. Humanized monoclonalantibodies are produced by transferring mouse complementaritydetermining regions from heavy and light variable chains of the mouseimmunoglobulin into a human variable domain, and then substituting humanresidues in the framework regions of the murine counterparts. The use ofantibody components derived from humanized monoclonal antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions. General techniques for cloning murine immunoglobulinvariable domains are described, for example, by Orlandi et al., Proc.Natl. Acad. Sci. USA 86:3833, 1989, which is hereby incorporated in itsentirety by reference. Techniques for producing humanized monoclonalantibodies are described, for example, by Jones et al., Nature 321:522,1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science239:1534, 1988; Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285,1992;Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singer et al., J. Immunol.150:2844, 1993, which are hereby incorporated by reference.

[0099] Antibodies of the invention also may be derived from humanantibody fragments isolated from a combinatorial immunoglobulin library.See, for example, Barbas et al., in: Methods: a Companion to Methods inEnzymology, Vol. 2, page 119, 1991; Winter et al., Ann. Rev. Immunol.12:433,1994, which are hereby incorporated by reference. Cloning andexpression vectors that are useful for producing a human immunoglobulinphage library can be obtained, for example, from STRATAGENE CloningSystems (La Jolla, Calif.).

[0100] In addition, antibodies of the present invention may be derivedfrom a human monoclonal antibody. Such antibodies are obtained fromtransgenic mice that have been “engineered” to produce specific humanantibodies in response to antigenic challenge. In this technique,elements of the human heavy and light chain loci are introduced intostrains of mice derived from embryonic stem cell lines that containtargeted disruptions of the endogenous heavy and light chain loci. Thetransgenic mice can synthesize human antibodies specific for humanantigens, and the mice can be used to produce human antibody-secretinghybridomas. Methods for obtaining human antibodies from transgenic miceare described by Green et al., Nature Genet. 7:13, 1994; Lonberg et al.,Nature 368:856, 1994; and Taylor et al., Int. Immunol. 6:579,1994, whichare hereby incorporated by reference.

[0101] The term “antibody” includes intact molecules as well asfragments thereof, such as Fab, (Fab′)₂, and Fv which are capable ofbinding the epitopic determinant. These antibody fragments retain someability to selectively bind with its antigen or receptor and are definedas follows:

[0102] (1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

[0103] (2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

[0104] (3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; (Fab′)₂, is a dimer of two Fab′ fragments held together bytwo disulfide bonds;

[0105] (4) Fv, defined as a genetically engineered fragment containingthe variable region of the light chain and the variable region of theheavy chain expressed as two chains; and

[0106] (5) Single chain antibody (“SCA”), defined as a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

[0107] Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York, 1988, incorporated herein by reference). Asused in this invention, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

[0108] Antibodies which bind to the EDG8 polypeptide of the inventioncan be prepared using an intact polypeptide or fragments containingsmall peptides of interest as the immunizing antigen. The polypeptide ora peptide used to immunize an animal can be derived from translated cDNAor chemical synthesis which can be conjugated to a carrier protein, ifdesired. Such commonly used carriers which are chemically coupled to thepeptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovineserum albumin (BSA), cmd tetanus toxoid. The coupled peptide is thenused to immunize the animal (e.g., a mouse, a rat, or a rabbit).

[0109] If desired, polyclonal or monoclonal antibodies can be furtherpurified, for example, by binding to and elution from a matrix to whichthe polypeptide or a peptide to which the antibodies were raised isbound. Those of skill in the art will know of various techniques commonin the immunology arts for purification and/or concentration ofpolyclonal antibodies, as well as monoclonal antibodies (See forexample, Coligan et al., Unit 9, Current Protocols in Immunology, WileyInterscience, 1991, incorporated by reference).

[0110] It is also possible to use the anti-idiotype technology toproduce monoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody.

[0111] Diagnostics

[0112] Further, the invention relates to a process for diagnosing adisease or a susceptibility to a disease (such as cancer, angiogenesisand inflammation that implicates S1P in pathophysiological states of thediseases) (Pyne and Pyne, Biochem J 349(Part 2):385, 2000) related toexpression or biological activity of EDG8 polypeptide comprising:

[0113] a) determining the presence or absence of mutation in thenucleotide sequence encoding said EDG8 polypeptide in the genome of saidsubject; and/or

[0114] b) analyzing for the presence or amount of the EDG8 polypeptideexpression in a sample derived from said subject.

[0115] This invention is also related to the use of the nucleic acidsencoding EDG8 as part of a diagnostic assay for detecting diseases orsusceptibility to diseases related to the presence of mutated G-proteincoupled receptor genes, such as EDG8 of SEQ ID NO:1. Such diseases arerelated to cell transformation, such as tumors and cancers.

[0116] Individuals carrying mutations in the human G-protein coupledreceptor gene may be detected at the DNA level by a variety oftechniques. Nucleic acids for diagnosis may be obtained from a patient'scells, such as from blood, urine, saliva, tissue biopsy and autopsymaterial. The genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR (Saiki et al., Nature 324:163,1986) prior to analysis. RNA or cDNA may also be used for the samepurpose. As an example, PCR primers complementary to the nucleic acidencoding the EDG8 polypeptide can be used to identify and analyze EDG8mutations. For example, deletions and insertions can be detected by achange in size of the amplified product in comparison to the normalgenotype. Point mutations can be identified by hybridizing amplified DNAto radiolabeled EDG8 receptor RNA or alternatively, radiolabeled EDG8receptor antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

[0117] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science 230:1242, 1985).

[0118] Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method (e.g., Cotton et al., PNAS USA, 85:4397,1985).

[0119] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

[0120] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations can also be detected by in situ analysis.

[0121] The sequences of the present invention are also valuable forchromosome identification (see Table-1). The sequence is specificallytargeted to and can hybridize with a particular location on anindividual human chromosome. Moreover, there is a current need foridentifying particular sites on the chromosome. Few chromosome markingreagents based on actual sequence data (repeat polymorphisms) arepresently available for marking chromosomal location. The mapping ofDNAs to chromosomes according to the present invention is an importantfirst step in correlating those sequences with genes associated withdisease.

[0122] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

[0123] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0124] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0125] Expression Vector

[0126] The invention refers further to a vector, preferably arecombinant DNA expression vector. In one embodiment, the inventionrelates to a DNA vector comprising a nucleic acid molecule consisting ofSEQ ID No. 1. The vector further comprises a polynucleotide elementwhich renders the vector suitable for its multiplication in procaryoticor eucaryotic cells and a DNA sequence as aforementioned coding for theamino acid sequence or a polynucleotide sequence for EDG8. The term“expression vector” refers to a plasmid, virus or other vehicle known inthe art that has been manipulated by insertion or incorporation of theEDG8 genetic sequences. This DNA element which renders the vectorsuitable for multiplication can be an origin of replication which worksin procaryotic or eucaryotic cells. An example for an origin ofreplication which works in procaryotic cells is the colE1 ori. Arecombinant vector needs further a selection marker for control ofgrowth of these organisms which harbor the vector. Suitable selectionmarkers include genes which protect organisms from antibiotics(antibioticum resistance) e.g. ampicillin, streptomycin, chloramphenicolor provide growth under compound deprived environmental conditions(auxotrophic growth conditions) when expressed as proteins in cells. Ina preferred embodiment of the invention for multiplication of the saidrecombinant vector the procaryotic cells are bacteria. In specialpreferred versions of the inventions the bacteria are in particularbacteria of Escherichia coli or of Bacillus spec. In a further preferredembodiment of the invention for the multiplication of the saidrecombinant vector the eucaryotic cells are cells of a cell line oryeast cells. In special preferred versions of the invention the cells ofthe cell line are cells of a CHO cell line.

[0127] Methods of expressing DNA sequences having eukaryotic or viralsequences in prokaryotes are well known in the art. Biologicallyfunctional viral and plasmid DNA vectors capable of expression andreplication in a host are known in the art. Such vectors are used toincorporate DNA sequences of the invention.

[0128] Thus, the nucleic acid sequence which encodes EDG8 can beoperatively linked to expression control sequences. “Operatively linked”refers to a juxtaposition wherein the components so described are in arelationship permitting them to function in their intended manner. Anexpression control sequence operatively linked to a coding sequence isligated such that expression of the coding sequence is achieved underconditions compatible with the expression control sequences. As usedherein, the term “expression control sequences” refers to nucleic acidsequences that regulate the expression of a nucleic acid sequence towhich it is operatively linked. Expression control sequences areoperatively linked to a nucleic acid sequence when the expressioncontrol sequences control and regulate the transcription and, asappropriate, translation of the nucleic acid sequence. Thus expressioncontrol sequences can include appropriate promoters, enhancers,transcription terminators, a start codon (i.e., ATG) in front of aprotein-encoding gene, splicing signal for introns, maintenance of thecorrect reading frame of that gene to permit proper translation of mRNA,and stop codons. The term “control sequences” is intended to include, ata minimum, components whose presence can influence expression, and canalso include additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences. Expressioncontrol sequences can include a promoter. By “promoter” is meant minimalsequence sufficient to direct transcription. Thus, the said recombinantDNA of the present invention could provide for a promotor element whichis operationally linked to a DNA sequence coding for the amino acidsequence or polynucleotide sequence of a EDG8 allowing transcription ofthe related RNA and/or expression of the related protein. Also includedin the invention are those promoter elements which are sufficient torender promoter-dependent gene expression controllable for cell-typespecific, tissue-specific, or inducible by external signals or agents;such elements may be located in the 5′ or 3′ regions of the gene. Thesepromotor elements can be taken in preferred versions of the inventionfrom procaryotic promoters or eucaryotic promoters. A procaryoticpromoter is characterized by its ability to induce transcription inprocaryotic organisms as a eucaryotic promoter is characterized by itsability to induce transcription in eucaryotic organisms. Bothprocaryotic and eucaryotic promoter elements can be preferred induciblepromoters or further preferred constitutive promoters (see e.g., Bitteret al., Methods in Enzymology 153:516-544, 1987). An inducible promoteris switched on only when a signal event is present. The signal can beborn by the organism's metabolism. Then it often consists of metabolicproducts, hormones, degradation products of macromolecules or othermetabolic derived substances. The signal can also be provided by theenvironment. Then it may consist of radiation, temperature or chemicalcompounds of the environment. A constitutive promoter needs no inductionfor activity. When cloning in bacterial systems, inducible promoterssuch as pL of bacteriophage, gamma, plac, ptrp, ptac (ptrp-lac hybridpromoter) and the like may be used. When cloning in mammalian cellsystems, promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) may be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences of the invention.

[0129] The invention includes further a host cell and a cell culturecomprised of said host cells. This host cell comprising at least onerecombinant DNA vector as mentioned before. “Host cells” are cells inwhich a vector can be propagated and its DNA expressed. The cell may beprokaryotic or eukaryotic. The term also includes any progeny of thesubject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term“host cell” is used. Methods of stable transfer, meaning that theforeign DNA is continuously maintained in the host, are known in theart. When the host cell is taken from procaryotic cells it preferablyconsists of a cell of a bacterium in particular of Escherichia coli orBacillus spec. When this host cell consists of a eucaryotic cell it ispreferred a cell of a cell line in particular a cell of a CHO cell line.

[0130] This host cell can be produced by transforming the said host cellby a recombinant DNA vector comprising a DNA sequence coding for anamino acid sequence or polynucleotide sequence of a EDG8. Thetransformation can take place by routine methods used in microbiology asfor example transformation of competent cells,Ca²⁺-phosphate-precipitation or electroporation. By “transformation” ismeant a genetic change induced in a cell following incorporation of newDNA (i.e., DNA exogenous to the cell). Where the cell is a mammaliancell, the genetic change is generally achieved by introduction of theDNA into the genome of the cell (i.e., stable).

[0131] By “transformed cell” is meant a cell into which (or into anancestor of which) has been introduced, by means of recombinant DNAtechniques, a DNA molecule encoding polypeptide of SEQ ID NO:1 or afragment thereof. Transformation of a host cell with recombinant DNA maybe carried out by conventional techniques as are well known to thoseskilled in the art. Where the host is prokaryotic, such as E. coli,competent cells which are capable of DNA uptake can be prepared fromcells harvested after exponential growth phase and subsequently treatedby the CaCl₂ method using procedures well known in the art.Alternatively, MgCl₂ or RbCl can be used. Transformation can also beperformed after forming a protoplast of the host cell if desired.

[0132] When the host is a eukaryote, such methods of transfection of DNAas calcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences of EDG8 of the invention. Anothermethod is to use a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein (see for example, EukaryoticViral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).

[0133] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

[0134] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification. Microbial cells employed in expression of proteinscan be disrupted by any convenient method, including freeze-thawcycling, sonication, mechanical disruption, or use of cell lysingagents, such methods are well know to those skilled in the art.

[0135] Isolation and purification of microbial expressed polypeptide, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

[0136] The EDG8 expressed polypeptides can be recovered and purifiedfrom recombinant host cells and cell cultures by methods includingammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatographyhydroxylapatite chromatography and lectin chromatography. Proteinrefolding steps can be used, as necessary, in completing configurationof the mature protein. Finally, high performance liquid chromatography(HPLC) can be employed for final purification steps.

[0137] In one embodiment, the invention provides substantially purifiedpolypeptide of SEQ ID No:2 or a fragment thereof. Preferably, EDG8translated polypeptide has an amino acid sequence set forth in SEQ IDNO:2. The term “substantially purified” as used herein refers to apolypeptide which is substantially free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.One skilled in the art can purify EDG8-polypeptide using standardtechniques for protein purification. The substantially pure polypeptidewill yield a single major band on a non-reducing polyacrylamide gel. Thepurity of the polypeptide can also be determined by amino-terminal aminoacid sequence analysis.

[0138] As explained above, the invention refers also to a proteinencoded by one of the DNA sequences as aforementioned. This protein hasactivity of a EDG8. Activity of EDG8 is meant the molecule is afunctional receptor for S1P, LPA, dHS1P and related lysophospholipidmediators. Such activity may be assayed using well known techniques inthe art. One such assay employs assessment of ability of Ca²⁺mobilization as described in FIG. 2. As described above, furtherincluded is production of a protein wherein first a host cell harboringa recombinant vector including a DNA sequence encoding for an amino acidsequence or a polynucleotide sequence for EDG8 is propagated in asuitable growth medium chosen from either media for bacteria oreucaryotic cells depending on the related host cell type. Thesepropagated cells are second harvested by common methods of biochemistryas centrifugation or filtration and processed to obtain crude cellextracts. These cell extracts third are purified subsequently by methodsused for protein purification as size exchange chromatography, ionexchange chromatography, affinity chromatography and others to gain theprotein of interest (EDG8 activity) separated from other compounds ofthe cell lysates.

[0139] The polypeptide of the invention may be expressed in a modifiedform, such as a fusion protein and may include not only secretionsignals, but also additional heterologous functional regions. Forinstance, a region of additional amino acids, particularly charged aminoacids, may be added to the N-terminus of the polypeptide to improvestability and persistence in the host cell, during purification, orduring subsequent storage and handling. Also, peptide moieties may beadded to the polypeptide to improve purification. Such regions may beremoved prior to final preparation of the peptide. Thus, in oneembodiment, the invention relates to a fusion protein comprising apolypeptide consisting of the amino acid sequence of SEQ ID NO. 2.Additionally, the fusion protein of the invention could include aminoacids of other members of the EDG family.

[0140] In one embodiment, the polypeptide of the present inventioncomprises the amino acid sequence of SEQ ID NO:2 and is encoded by thenucleotide sequence of SEQ ID NO:1. However, the polypeptide of theinvention can be varied without significant effect on the structure orfunction of the molecule.

[0141] Minor modifications of the EDG8 primary nucleotide sequences mayresult in proteins which have substantially equivalent activity ascompared to the unmodified counterpart polypeptide described herein.Such modifications may be deliberate, as by site-directed mutagenesis,or may be spontaneous. All of the polypeptides produced by thesemodifications are included herein as long as the biological activity ofthe EDG8 still exists.

[0142] The polypeptide of the present invention also includes fragmentsand variants of SEQ ID NO:2. “Variant” when referring to the polypeptideof SEQ ID NO:2, means polypeptides which retain essentially the samebiological function or activity as a polypeptide comprising the fulllength SEQ ID NO:2.

[0143] A “fragment” is a segment of SEQ ID NO:2 that comprisescontiguous amino acids.

[0144] The variant of the polypeptide SEQ ID NO:2 may be (i) one inwhich one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptides are fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptides. Such variants are deemed to bewithin the scope of those skilled in the art from the teachings herein.

[0145] The polypeptides of the present invention include the polypeptideof SEQ ID NO:2 as well as polypeptides which have at least about 70%similarity to the polypeptide of SEQ ID NO:2 and more preferably aboutat least a 90% similarity to the polypeptide of SEQ ID NO:2 and stillmore preferably at least about a 95% similarity to the polypeptide ofSEQ ID NO:2 and also includes fragments of such polypeptides with suchportion of the polypeptide generally containing about at least 8consecutive amino acids and preferably about at least 30 to 50consecutive amino acids.

[0146] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide. This can be done manually (using mathematical calculations)or with a computer program, such as the Wisconsin package version10.1-Unix (Genetics Computer Group (GCG), Madison, Wis.).

[0147] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis, therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments also may be used to generate antibodies, as described above.

[0148] In addition, the invention relates to a method for identifyingcompounds which bind to EDG8 polypeptide comprising:

[0149] a) contacting a cell comprising the expression system or a partof such a cell with a candidate compound; and

[0150] b) assessing the ability of said candidate compound to bind tosaid cells.

[0151] Preferably, the method for identifying compounds further includesdetermining whether the candidate compound effects a signal generated byactivation of the EDG8 polypeptide at the surface of the cell, wherein acandidate compound which effects production of said signal is identifiedas an agonist.

[0152] In another embodiment of the invention, the method foridentifying compounds further includes determining whether the candidatecompound effects a signal generated by activation of the EDG8polypeptide at the surface of the cell, wherein a candidate compoundwhich effects production of said signal is identified as an antagonist.

[0153] Screening and Uses as Therapeutics

[0154] The present invention also relates to a method for determiningwhether a ligand not known to be capable of binding to a G-proteincoupled receptor can bind to such receptor which comprises contacting amammalian cell which expresses a G-protein coupled receptor with theligand under conditions permitting binding of ligands to the G-proteincoupled receptor, detecting the presence of a ligand which binds to thereceptor and thereby determining whether the ligand binds to theG-protein coupled receptor. The systems hereinabove described fordetermining agonists and/or antagonists may also be employed fordetermining ligands which bind to the receptor. In the preferredembodiment, the receptor is EDG8.

[0155] In general, antagonists for G-protein coupled receptors which aredetermined by screening procedures may be employed for a variety oftherapeutic purposes. For example, such antagonists have been employedfor treatment of hypertension, angina pectoris, myocardial infarction,ulcers, asthma, allergies, psychoses, depression, migraine, vomiting,stroke, eating disorders, migraine headaches, cancer and benignprostatic hypertrophy.

[0156] Agonists for G-protein coupled receptors are also useful fortherapeutic purposes, such as the treatment of asthma, Parkinson'sdisease, acute heart failure, hypotension, urinary retention, andosteoporosis.

[0157] Examples of G-protein coupled receptor antagonists include anantibody, or in some cases an oligonucleotide, which binds to theG-protein coupled receptor but does not elicit a second messengerresponse such that the activity of the G-protein coupled receptor isprevented. Antibodies include anti-idiotypic antibodies which recognizeunique determinants generally associated with the antigen-binding siteof an antibody. Potential antagonists also include proteins which areclosely related to the ligand of the G-protein coupled receptor, i.e. afragment of the ligand, which have lost biological function and whenbinding to the G-protein coupled receptor, elicit no response.

[0158] The invention also relates to an agonist or antagonist identifiedby such methods.

[0159] In another special embodiment of the invention, the methodfurther includes contacting said cell with a known agonist for said EDG8polypeptide; and determining whether the signal generated by saidagonist is diminished in the presence of said candidate compound,wherein a candidate compound which effects a diminution in said signalis identified as an antagonist for said EDG8 polypeptide. The knownagonist is for example S1P, LPA and/or dHS1P. The invention also relatesto an antagonist identified by the method.

[0160] A compound can affect EDG8 by either stimulating or inhibitingEDG8 activity. An antagonist is a compound that directly or indirectly“inhibits” a signal generated by activation of the EDG8 polypeptide atthe surface of the cell. An agonist is a compound that directly orindirectly “stimulates” a signal generated by activation of the EDG8polypeptide.

[0161] Potential antagonists to the EDG8 polypeptides of the presentinvention include an antibody against the EDG8 polypeptides, or in somecases, an oligonucleotide, which bind to the EDG8 polypeptides and alterits conformation.

[0162] Potential antagonists also include antisense constructs producedby antisense technology. Antisense technology controls gene expressionthrough triple-helix formation, etc. The number of EDG8 may be reducedthrough antisense technology, which controls gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes for themature polypeptides of the present invention, is used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription (triple helix—see Lee etal., Nucl. Acids Res., 6:3073, 1979); Cooney et al, Science, 241:456,1988); and Dervan et al., Science, 251: 1360, 1991), thereby preventingtranscription and the production of the EDG8 polypeptides. The antisenseRNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into the EDG8 polypeptides(antisense—Okano, J. Neurochem., 56:560, 1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.1988). The antisense constructs can be delivered to cells by proceduresknown in the art such that the antisense RNA or DNA may be expressed invivo.

[0163] The antagonist or agonist compounds may be employed incombination with a suitable pharmaceutical carrier. Such compositionscomprise a therapeutically effective amount of the compound, and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

[0164] The invention in addition, relates to a method of preparing apharmaceutical composition comprising:

[0165] a) identifying a compound which is an agonist or an antagonist ofEDG8,

[0166] b) preparing the compound, and

[0167] c) optionally mixing the compound with suitable additives.

[0168] The invention also relates to a pharmaceutical compound preparedby such a process.

[0169] The invention relates to a pharmaceutical, comprising as activeingredient for example such identified compound, an EDG8 polypeptide ora polynucleotide encoding for EDG8 or a part thereof.

[0170] In particular, the invention relates to a pharmaceutical, thatcan be used for the prevention and/or treatment of diseases associatedwith EDG8/S1P signal transduction, for example diseases associated withendothelial dysfunction such as for example Atheriosclerosis, Shoke,Hypertonie, coronary syndroms, cancer, thrombolylic diseases, affectedwound healing and diseases accompanied by increased cell death. Inanother aspect of the invention, such pharmaceutical can be used for theprevention and/or treatment of diseases associated with a dysregulationof angiogenesis, such as for example tumor growth, rheumatical arthritisand diabetic setinopathy.

[0171] The study, reported about the cloning, chromosomal mapping,tissue expression and functional identification as a receptor for S1P ofa novel GPCR, EDG8, the fifth functional receptor for sphingosine1-phosphate.

[0172] In an effort to identify new G-protein coupled receptors of theEDG-family a database search with alignments of the currently known 18members of this receptor family was performed, comprising human EDG1-7sequences up to the putative EDGs from Xenopus and Zebra-fish. Amultiple alignment of these sequences was created by CLUSTALW and usedin a PSI-BLAST search to scan translated versions of human genomic DNAsequences, which were publicly available in the different EMBL sections.For translation of DNA into protein sequences, individual protein fileswithin two respective STOP-codon were created and all proteins shorterthan 50 amino acids were ignored. As the majority of GPCRs is unsplicedsearching for GPCRs within genomic sequences should bring about novelreceptor proteins.

[0173] Performing a PSI-BLAST search, the various cDNAs and genomiccontigs, respectively, for the human EDG1-7 receptors were identified,and an additional genomic hit, highly homologous to human EDG5 (51%homology), termed EDG8. The nucleotide and amino acid sequence of thenew putative GPCR are depicted in FIG. 1A. Hydropathy analysis(hydrophobicity plot not shown) suggests a seven transmembrane proteinwith three alternating extra- and intracellular loops, assumed to be theheptahelix structure common to GPCRs.

[0174] To shed more light on the relationships involved in the molecularevolution of the EDG-receptor family, a grow tree phylogram wasconstructed using the neighbor joining method (Genetic Computer Group(GCG), Madison, Wis. (FIG. 1B) Comparison of amino acid sequences).According to this phylogenetic tree, the human EDG-family can be dividedinto two distinct groups: EDG1, 3, 5 and 6 belonging to one, EDG2, 4 and7 belonging to the other group. These two groups are discriminatedfurther by their preference for different lipid ligands: EDG1, 3, 5, 6are preferentially stimulated by sphingosin 1-phosphate (S1P) (Yatomi etal., J Biochem (Tokyo) 12:969, 1997; Lee et al., Science 279:1552, 1998;Lee et al., J Biol Chem 273:22105,1998; Ancellin and Hla, J Biol Chem274:18997,1999; Yamazaki et al., Biochem Biophys Res Commun 268:583,2000; Van Brocklyn et al., Blood 95:2624, 2000), EDG2, 4 and 7 bylysophosphatidic acid (LPA) (Hecht et al., 1996; An et al., J Biol Chem273:7906, 1998; Im et al., Mol Pharmacol 57:753, 2000). The newlyidentified EDG8 exhibited highest similarity (86.8% amino acid identity)to the rat nrg1-protein (FIG. 1B), a GPCR recently cloned byEST-expression profiling from a rat PC12 cell library (Glickman et al.,Mol Cell Neuroscience 14:141, 1999), which probably represents the rathomologue of human EDG8. In the report of Glickman et al., however, theauthors did not address the question of the activating ligand of thisreceptor. The high similarity between EDG8 and the known sphingosin1-phosphate (S1P) receptors EDG1, 3 and 5 (48-51%) (FIG. 1C) led to testthe hypothesis that EDG8 may be a functional S1P-receptor.

[0175] In testing for S1P receptor activity, the EDG8 cDNA wasintroduced into chinese hamster ovary (CHO) cells by transienttransfection. CHO cells were chosen as they exhibit minimal responses tosphingosin 1-phosphate in concentrations up to 1 μM but respond to S1Pafter transfection with the S1P preferring receptors EDG 1, 3 and 5(Okamoto et al., J Biol Chem 273:27104,1998; Kon et al., J Biol Chem274:23940, 1999). To test functional receptor activity the mobilizationof [Ca²⁺]_(i) was monitored for three reasons:

[0176] 1) S1P has been reported to increase Ca²⁺ in many cell types(Ghosh et al., 1990; Zang et al., 1991; Durieux et al., Am J Physiol264:C1360, 1993; Chao et al., J Biol Chem 269:5849, 1994; Gosh et al., JBiol Chem 269:22628, 1994; Mattie et al., J Biol Chem 269:3181,1994;Meyer zu Heringdorf et al., Naunyn-Schmiedeberg's Arch Pharmacol354:397, 1997; Okajima et al., FEBS Lett 379:260, 1996; van Koppen etal., J Biol Chem 271:2082, 1996; Törnquist et al., Endocrinology138:4049, 1997; Yatomi et al., J Biochem (Tokyo) 12:969, 1997; Noh etal., J Cell Physiol 176:412,1998; An et al., Mol Pharmaco 55:787,1999).

[0177] 2) the identification of EDG1, 3, 5 and 6 as receptors for S1Phas provided the molecular basis for a GPCR mediated mechanism and thereceptors are known to mediate intracellular Ca²⁺-release through eitherPTX-sensitive Gα_(i) proteins or the PTX-insensitive Gα_(q/11) pathway(Okamoto et al., J Biol Chem 273:27104,1998; Kon et al., J Biol Chem274:23940,1999; Gonda et al., Biochem J 337:67, 1999).

[0178] 3) all currently known S1P-responding EDG-receptors (except EDG6)are present in endothelial cells (A. Niedernberg et al., submitted), inwhich intracellular Ca²⁺ release is a major pathway in the generation ofNO, an important factor in vascular biology. Thus, identification of thecomplete set of S1P receptors, involved in intracellular Ca²⁺mobilization could help clarify the role of the individual subtypes inendothelial cell signaling.

[0179]FIG. 2 depicts measurement of the intracellular Ca²⁺concentration, mediated by S1P via the putative S1P receptor EDG8. Forsake of comparison, the S1P-receptors EDG1, 3, 5, and 6, which have beenreported to mobilize [Ca²⁺]_(i), were included. [Ca²⁺]_(i) were recordedas real time measurements using the Fluorescence plate imaging reader(FLIPR, Molecular Devices). Initially, CHO cells transfected with emptyvector DNA were stimulated with different concentrations of S1P (10,100, 1000 nM). None of the applied S1P concentrations was capable ofeliciting significant rises in intracellular Ca²⁺ (FIG. 2A), suggestingthat S1P receptors are not expressed in CHO cells or, if expressed, areunable to signal via the endogeneous Gα_(q) pathway. To address thisissue, the G protein chimera Gα_(qi5), which confers onto Gi coupledreceptors the ability to stimulate the Gq pathway, and Gα₁₆, which linksGi- and Gs coupled receptors to PLCβ and subsequent intracellularCa²⁺-mobilization were used. Upon stimulation with S1P, G_(qi5)- andG₁₆-transfected CHO cells did not give rise to significant increases in[Ca²⁺]_(i) (FIG. 2A). However, transient transfection of CHO-cells withthe cDNAs coding for the EDG1, 3 and 5 receptor conferredS1P-responsiveness to the cells: it was confirmed that EDG1, 3 and 5mobilize [Ca²⁺]_(i) in response to S1P (FIGS. 2B, C, D) (Kon et al., JBiol Chem 274:23940, 1999). As already known for a large number ofGq-coupled receptors, coexpression of Gα_(q) augments the EDG1 and5-mediated Ca²⁺-response as compared with the Ca²⁺ signal induced bystimulation of endogeneous Gα_(q). In case of EDG3, additionalexogeneously added Gα_(q) did not further improve the signal intensity.These results are in agreement with the findings reported by Kon et al.(J Biol Chem 274:23940, 1999), who showed that the EDG3-subtype causesthe most robust enhancement of intracellular Ca²⁺.

[0180] In case of EDG6, Yamazaki et al. (Biochem Biophys Res Commun268:583, 2000) obtained an S1P-induced mobilization of [Ca²⁺]_(i) but inthis study, investigators failed to detect a significant Ca²⁺ increaseabove basal levels in the absence of any cotransfected G-protein asubunit (FIG. 2E). The reason for this discrepancy could be the cellularbackground (CHO cells in this study vs. K562 cells in Yamazaki et al.,Biochem Biophys Res Commun 268:583, 2000), as they reported a pertussistoxin (PTX)-sensitive Ca²⁺-response, indicating the involvement ofGi-type G-proteins. In this case the Ca²⁺ signal would be elicited byβγ, released from activated Gα_(i)βγ heterotrimers. The Gα_(i)-inducedCa²⁺ signals are known to be much smaller in intensity as compared withthe Ca²⁺ signals induced by bona-fide Gq-linked receptors (Kostenis etal., J Biol Chem 272:19107,1997). It may be that detection of such[Ca²⁺]_(i) concentrations is beyond the sensitivity of the FLIPR system.

[0181] EDG8 did not release [Ca²⁺]_(i) when stimulated with S1P (10,100, and 1000 nM) (FIG. 2F), but gained the ability to mobilize Ca²⁺upon cotransfection with Gα₁₆, a G-protein a subunit, known to coupleGPCRs from different functional classes to the Gq-PLCβ pathway orGα_(qi5), a mutant G-protein a subunit that confers onto Gi-linkedreceptors the ability to stimulate Gq (Conklin et al., 1993). Theseresults show that EDG8 is a functional receptor for S1P and thatEDG8-induced Ca²⁺ responses are due to a non-Gq pathway, probably theactivation of phospholipase Cβ2 by βγ subunits of the Gi proteins.Furthermore, these results provide additional evidence that theS1P-preferring EDG-receptors couple differentially to the Gq and Gipathways: EDG3 ist the most potent Ca²⁺-mobilizing receptor andoverexpression of Gα_(q) does not further improve Ca²⁺ signalling; EDG1and 5 induce moderate Ca²⁺-increases, that can be significantly improvedby cotransfection of Gα_(q) or a chimeric Gα_(qi5) protein;EDG8-mediated Ca²⁺-responses require cotransfection of Gα_(qi5) or Gα₁₆.

[0182] To check whether the EDG8 receptor also reacts to relatedlysophospholipid mediators, the inventors examined the abilities oflysophosphatidic acid (LPA), dihydrosphingosin 1-phosphate (dHS1P),sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC) toincrease intracellular Ca²⁺ in CHO cells transiently transfected withthe EDG8 receptor and the G-protein a subunits Gα₁₆ and Gα_(qi5)(FIG.3). Besides S1P, which was the most potent activator of EDG8, LPA anddHS1P evoked [Ca²⁺]_(i) increases in concentrations of 100 and 1000 nM.SPC and LPC, respectively, failed to generate any significant responsein concentrations up to 1 μM. These data show that EDG8 is a S1Ppreferring receptor, but also responds to related phospholipids likedHS1P or LPA, as has also been reported for EDG1, which is a highaffinity receptor for S1P and a low affinity receptor for LPA (Lee etal., J Biol Chem 273:22105,1998). Therefore, EDG8 receptor has thecharacteristic functionality to respond to S1P and related phospholipidslike DMS 1P or LPA. The response to S1P and other related phospholipidescan for example be determined as described in Example 3. Cellscontaining the respective Gα can be obtained as described in Example 2.

[0183] Next, the expression pattern of the EDG8 gene in human tissueswas investigated by Northern blot analysis (FIG. 4). Tissues positivefor EDG8 RNA were skeletal muscle, heart and kidney, lower abundance ofRNA was seen in liver and placenta, no signal was detected in brain,thymus, spleen, lung and peripheral blood leukocytes. In all tissues asingle RNA transcript of 5.5 kb was observed after hybridization with aDIG-labelled EDG8 antisense RNA probe. EDG8 exhibits highest similarityto the rat nrg1-GPCR (Glickman et al., Mol Cell Neuroscience14:141,1999) with an amino acid identity of 86.8% (FIG. 1B) suggestingthat it may be the human homolog of the rat nrg1 protein. However, theexpression pattern of human EDG8 is quite different from the ratnrg1-receptor, which is found almost exclusively in brain (Glickman etal., Mol Cell Neuroscience 14:141,1999). This finding suggests that EDG8may represent a closely related but entirely different receptor fromnrg1, rather than the human homolog. Never the less, it does not ruleout the possibility that EDG8 and nrg1 are homologs with entirelydifferent, species-dependent expression patterns.

[0184] As the first member of the EDG-family of GPCRs—EDG1—wasoriginally cloned as an endothelial differentiation gene fromphorbol-myristic-acetate-treated differentiating human endothelial cells(Hla and Maciag, J Biol Chem 265:9308, 1990) and subsequently clonedfrom a human umbilical vein endothelial cell library exposed to fluidshear stress as an upregulated gene it is reasonable to assume that EDGreceptors play an important role in the regulation of endothelialfunction. Therefore, the presence of EDG8 transcripts in several humanendothelial cell lines was analyzed. RT-PCR analysis of human umbilicalvein endothelial cells (HUVECs), human coronary artery endothelial cells(HCAECs), human microvascular endothelial cells of the lung (HMVEC-L)and human pulmonary artery endothelial cells (HPAEC) revealed EDG8expression in all cell lines tested (FIG. 5A). In FIG. 5B it is shownthat EDG8 specific primers indeed solely amplify EDG8 sequences and noneof the related EDG1-7 sequences. These findings suggest that thepresence of EDG8 in different peripheral organs may be due to itslocalization in endothelial cells; it does not rule out, however, thatEDG8 transcripts occur in cell types other than endothelial cells.

[0185] The expression of EDG8 in addition to EDG1, 3, and 5 (Rizza etal., Laboratory Investigation 79:1227, 1999) in HUVECs and several otherendothelial cell lines is intriguing in view of all the reportsregarding S1P effects on endothelial cell signalling. Hisano et al.(Blood 93:4293, 1999) reported that S1P protects HUVECs from apoptosisinduced by withdrawal of growth factors and stimulates HUVEC DNAsynthesis; the authors derived a model for cell-cell interactionsbetween endothelial cells and platelets but the S1P-receptor responsiblefor HUVEC-protection of apoptosis could not be identified. Rizza et al.(Laboratory Investigation 79:1227, 1999) reported that SIP plays a rolein endothelial cell leukocyte interaction in that S1P induces expressionof cell adhesion molecules in human aortic endothelial cells, allowingmonocytes and neutrophils to attach. These effects were blocked bypertussis toxin, suggesting the involvement of a Gi-coupled S1Preceptor. The responsible S1P-receptor subtype, however, could not beidentified and the EDG8 receptor was not included at the time of thisstudy. Expression profiling of all EDG receptors in individual celllines and the use of EDG receptor subtype selective compounds willclearly be necessary to help determine the role of the individual S1Preceptors in endothelial cell signalling mechanisms.

[0186] Finally, the mapping of EDG receptors in genomic sequencesallowed to derive the chromosomal localization for four genes of thisfamily (Table 1). Interestingly, so far, four EDG-receptors includingEDG8 are located on chromosome 19. In addition, the genomic sequenceallowed the determination of the structure of the genes: theS1P-preferring receptors EDG1, 3, 5 and 8 are intronless as opposed tothe LPA-preferring subtypes 2, 4 and 7, that contain an intron in theopen reading frame in TMVI. These data suggest that in addition to theactivating ligand and the degree of homology, the two subclasses oflysophospholipid receptors can be discriminated further by their genomicstructure. The genomic structure of new potential EDG/LPA-receptorfamily members may also help predict the nature of the activating lipidligand.

[0187] In conclusion, a new member of the EDG-family of G-proteincoupled receptor, human EDG8, was isolated. This receptor functions as acellular receptor for sphingosine 1-phosphate. EDG8 could exclusively bedetected in peripheral tissues like skeletal muscle, heart and kidneyand several human endothelial cell lines. It is conceivable that theexpression in endothelial cells may account for the broad tissuedistribution of this receptor. The existence of at least eightEDG-receptors for lysophospholipids suggests that receptor subtypeselective agonists and antagonists will essentially be necessary for abetter understanding of the biology of lysophospholipids and theirrespective receptors. TABLE 1 Chromosomal localization, gene structureand accession number of the respective EDG genomic clones. Mapping ofEDG receptors in genomic sequences allowed to derive a chromosomalassignment for EDG1, 2, 4-8. The chromosomal localization of EDG3 wasobtained from Yamaguchi et al. (1996). Genomic sequences also revealedEDG1, 3, 5, 6 and 8 to be unspliced as opposed to EDG2, 4 and 7, whichcontain an intron in their open reading frame (ORF). Chromosomallocalization according BAC EDG spliced/unspliced in ORF accessionnumber: EDG1 1p21.1-21.3 unspliced AL161741 EDG2 9q31.1-32/ /18p11.3spliced AL157881/ /AP000882 EDG3 9q22.1-q22.2 unspliced EDG4 19p12spliced NT_000939 EDG5 19 unspliced AC011511 EDG6 19p13.3 unsplicedAC011547 EDG7 1p22.3-31.2 spliced AL139822 EDG8 19 unspliced AC011461

EXAMPLE 1 Molecular Cloning of the Human EDG8 Receptor

[0188] As the putative human EDG8 sequence is intronless, the receptorwas cloned from human genomic DNA (CLONTECH, Palo Alto, Calif.,94303-4230) via polymerase chain reaction (PCR). PCR conditions,established to amplify the EDG8 sequence were 94° C., 1 min followed by35 cycles of 94° C., 30sec, 68° C., 3 min, using GC-Melt Kit (CLONTECH,Palo Alto, Calif.). Primers designed to amplify the EDG8 sequencecontained a HindIII site in the forward, and a EcoRI site in the reverseprimer, respectively. The 1197 bp PCR product was cloned into thepCDNA3.1 (+) mammalian expression vector (Invitrogen, Carlsbad, Calif.)and sequenced in both directions.

EXAMPLE 2 Cell Culture and Transfection

[0189] CHO-K1 cells were grown in basal ISCOVE medium supplemented with10% fetal bovine serum at 37° C. in a humidified 5% CO₂ incubator. Fortransfections, 2×10⁵ cells were seeded into 35-mm dishes. About 24 hrlater cells were transiently transfected at 50-80% confluency with theindicated receptor and G-protein constructs (1 μg of plasmid DNA each)using the Lipofectamine transfection reagent and the supplied protocol(GIBCO). 18-24 hr after transfection cells were seeded into 96wellplates at a density of 50.000 cells per well and cultured for 18-24additional hr until used in the functional FLIPR assays.

[0190] The cDNA for Gα16 was cloned from TF1 cells by RT-PCR and ligatedinto the pCDNA1.1 mammalian expression vector (Invitrogen). Murine wildtype Gαq was cloned from cells by RT-PCR and inserted into theBamHI-NsiI-sites of pCDNA1.1. To create the C-terminally modifiedGα_(qi5) subunit, in which the last five aa of wt Gαq were replaced withthe correspoding Gα_(i) sequence, a 175-bp BgIII-NsiI fragment wasreplaced, in a two piece ligation, with a synthetic DNA fragment,containing the desired codon changes. The correctness of all PCR-derivedsequences was verified by sequencing in both directions.

EXAMPLE 3 Fluorometric Imaging Plate Reader (FLIPR) Assay

[0191] Twenty-four hours after transfection, cells were splitted into96-well, black-wall microplates (Corning) at a density of 50,000 cellsper well. 18-24 hr later, cells were loaded with 95μl of HBSS containing20 mM Hepes, 2.5 mM probenecid, 4 μM fluorescent calcium indicator dyeFluo4 (Molecular Probes) and 1% fetal bovine serum for 1 h (37° C., 5%CO₂). Cells were washed three times with HBSS containing 20 mM Hepes and2.5 mM probenecid in a cell washer. After the final wash, the solutionwas aspirated to a residual volume of 100 μl per 96 well. Lipid ligandswere dissolved in DMSO as 2 mM stock solutions (treated with ultrasoundwhen necessary) and diluted at least 1:100 into HBSS containing 20 mMHEPES, 2.5 mM probenecid and 0.4 mg/ml fatty acid free bovine serumalbumine. Lipids were aliquoted as 2× solutions into a 96 well plateprior to the assay. The fluorometric imaging plate reader (FLIPR,Molecular Devices) was programmed to transfer 100 μl from each well ofthe ligand microplate to each well of the cellplate and to recordfluorescence during 3 min in 1 second intervals during the first minuteand 3 second intervals during the last two minutes. Total fluorescencecounts from the 18-s to 37-s time points are used to determine agonistactivity. The instrument software normalizes the fluorescent reading togive equivalent initial readings at time zero.

EXAMPLE 4 Northern Blot Analysis

[0192] Human multiple tissue Northern blots were purchased from CLONTECH(Palo Alto, Calif., 94303-4230, USA). Antisense RNA probes weregenerated by subcloning nucleotides 279-1197 of the coding region intothe Bam HI-Eco RI sites of the expression vector PSPT18 (RocheDiagnostics, Mannheim, Germany) and subsequent random priming with aDIG-RNA Labeling kit (Roche Diagnostics, Mannheim, Germany), using T7RNA polymerase. Hybridization was carried out at 68° C. for 16 h inhybridization buffer (Dig Easy Hyb Roche Diagnostics, Mannheim,Germany). Each blot was washed , blocked and detected as indicated inthe standard protocol with the DIG Wash and Block Buffer set (RocheDiagnostics, Mannheim, Germany) and treated with 1 ml CSPDready-to-use(Roche Diagnostics, Mannheim, Germany) for 15 min, 37° C.and developed for 5 min on the Lumiimager (Roche). Finally, each blotwas stripped (50% formamide, 5% SDS, 50 mM Tris/HCl pH 7.5; 80° C., 2× 1hour) and rehybridized with a GAPDH anti-sense RNA probe as an internalstandard.

EXAMPLE 5 RNA Extraction and RT-PCR

[0193] RNA was prepared from different endothelial cell lines (HUVECS,HCAEC, HMVEC-L, HPAEC) using the TRIzol reagent (Hersteller, Lok.).Briefly, for each endothelial cell line, cells of a subconfluent 25 cm2tissue culture flask were collected in 2.5 ml TRIzol and total RNAs wereextracted according to the supplied protocol. The purity of the RNApreparation was checked by veryfying the absence of genomic DNA. Analiquot of RNA, corresponding to ˜5 μg, was used for the cDNA generationusing MMLV reverse transcriptase and the RT-PCR kit from STRATAGENE.RT-PCR was carried out in a volume of 50 μl, the RT-PCR conditions wereset to 65° C. for 5 min, 15 min at RT, 1 hour at 37° C., 5 min at 90°C., chill on ice. The cDNA templates for the PCR reactions (35 cycles of94° C. for 30 sec, 68° C. for 3 min) were the reverse transcribedproducts of RNAs isolated from human endothelial cell lines (HUVECS,HCAEC, HMVEC-L, HPAEC). Typically, 1-5 μl of reverse transcribed cDNAswere used as templates for the PCR reactions.

EXAMPLE 6 Sources of Materials

[0194] 1-oleoyl-LPA, sphingosin 1-phosphate (S1P), dihydrosphingosin1-phosphate (dHS1P), lysophosphatidylcholine (LPC),sphingosylphosphorylcholine (SPC) and fatty acid free BSA were fromSIGMA (P.O. Box 14508, St. Louis, Mo. 63178). CHO-K1 cells were obtainedfrom the American Type culture collection (ATCC, Manassas, Va.), cellculture media and sera from GIBCO BRL (Gaithersburg, Md.), the Cafluorescent dye FLUO4 and pluronic acid from Molecular devices(Sunnyvale Calif. 94089-1136,USA) human northern blot membrane fromCLONTECH (1020 East Meadow Circle, Palo Alto, Calif. 94303-4230, USA.),commercially available cDNAs (heart, fetal heart, left atrium, leftventricle, kidney, brain, liver, lung, aorta) from Invitrogen,oligonucleotides from MWG-Biotech AG (Ebersberg, Germany), the RT-PCRkit from SIGMA, the GC-melt PCR kit from Clontech (Palo Alto, Calif.),the expression plasmid pcDNA3.1 for EDG8 and pCDNA1.1 for expression ofG-protein α subunits from Invitrogen (Carlsbad, Calif. 92008), competentDH5α from GIBCO and MC 1063 from Invitrogen.

[0195] All citations, including patents, patent applications, journalarticles, books and other publications are herein incorporated byreference in their entirety.

1 9 1 1197 DNA Homo sapiens CDS (1)..(1194) 1 atg gag tcg ggg ctg ctgcgg ccg gcg ccg gtg agc gag gtc atc gtc 48 Met Glu Ser Gly Leu Leu ArgPro Ala Pro Val Ser Glu Val Ile Val 1 5 10 15 ctg cat tac aac tac accggc aag ctc cgc ggt gcg cgc tac cag ccg 96 Leu His Tyr Asn Tyr Thr GlyLys Leu Arg Gly Ala Arg Tyr Gln Pro 20 25 30 ggt gcc ggc ctg cgc gcc gacgcc gtg gtg tgc ctg gcg gtg tgc gcc 144 Gly Ala Gly Leu Arg Ala Asp AlaVal Val Cys Leu Ala Val Cys Ala 35 40 45 ttc atc gtg cta gag aat cta gccgtg ttg ttg gtg ctc gga cgc cac 192 Phe Ile Val Leu Glu Asn Leu Ala ValLeu Leu Val Leu Gly Arg His 50 55 60 ccg cgc ttc cac gct ccc atg ttc ctgctc ctg ggc agc ctc acg ttg 240 Pro Arg Phe His Ala Pro Met Phe Leu LeuLeu Gly Ser Leu Thr Leu 65 70 75 80 tcg gat ctg ctg gca ggc gcc gcc tacgcc gcc aac atc cta ctg tcg 288 Ser Asp Leu Leu Ala Gly Ala Ala Tyr AlaAla Asn Ile Leu Leu Ser 85 90 95 ggg ccg ctc acg ctg aaa ctg tcc ccc gcgctc tgg ttc gca cgg gag 336 Gly Pro Leu Thr Leu Lys Leu Ser Pro Ala LeuTrp Phe Ala Arg Glu 100 105 110 gga ggc gtc ttc gtg gca ctc act gcg tccgtg ctg agc ctc ctg gcc 384 Gly Gly Val Phe Val Ala Leu Thr Ala Ser ValLeu Ser Leu Leu Ala 115 120 125 atc gcg ctg gag cgc agc ctc acc atg gcgcgc agg ggg ccc gcg ccc 432 Ile Ala Leu Glu Arg Ser Leu Thr Met Ala ArgArg Gly Pro Ala Pro 130 135 140 gtc tcc agt cgg ggg cgc acg ctg gcg atggca gcc gcg gcc tgg ggc 480 Val Ser Ser Arg Gly Arg Thr Leu Ala Met AlaAla Ala Ala Trp Gly 145 150 155 160 gtg tcg ctg ctc ctc ggg ctc ctg ccagcg ctg ggc tgg aat tgc ctg 528 Val Ser Leu Leu Leu Gly Leu Leu Pro AlaLeu Gly Trp Asn Cys Leu 165 170 175 ggt cgc ctg gac gct tgc tcc act gtcttg ccg ctc tac gcc aag gcc 576 Gly Arg Leu Asp Ala Cys Ser Thr Val LeuPro Leu Tyr Ala Lys Ala 180 185 190 tac gtg ctc ttc tgc gtg ctc gcc ttcgtg ggc atc ctg gcc gct atc 624 Tyr Val Leu Phe Cys Val Leu Ala Phe ValGly Ile Leu Ala Ala Ile 195 200 205 tgt gca ctc tac gcg cgc atc tac tgccag gta cgc gcc aac gcg cgg 672 Cys Ala Leu Tyr Ala Arg Ile Tyr Cys GlnVal Arg Ala Asn Ala Arg 210 215 220 cgc ctg ccg gca cgg ccc ggg act gcgggg acc acc tcg acc cgg gcg 720 Arg Leu Pro Ala Arg Pro Gly Thr Ala GlyThr Thr Ser Thr Arg Ala 225 230 235 240 cgt cgc aag ccg cgc tcg ctg gccttg ctg cgc acg ctc agc gtg gtg 768 Arg Arg Lys Pro Arg Ser Leu Ala LeuLeu Arg Thr Leu Ser Val Val 245 250 255 ctc ctg gcc ttt gtg gca tgt tggggc ccc ctc ttc ctg ctg ctg ttg 816 Leu Leu Ala Phe Val Ala Cys Trp GlyPro Leu Phe Leu Leu Leu Leu 260 265 270 ctc gac gtg gcg tgc ccg gcg cgcacc tgt cct gta ctc ctg cag gcc 864 Leu Asp Val Ala Cys Pro Ala Arg ThrCys Pro Val Leu Leu Gln Ala 275 280 285 gat ccc ttc ctg gga ctg gcc atggcc aac tca ctt ctg aac ccc atc 912 Asp Pro Phe Leu Gly Leu Ala Met AlaAsn Ser Leu Leu Asn Pro Ile 290 295 300 atc tac acg ctc acc aac cgc gacctg cgc cac gcg ctc ctg cgc ctg 960 Ile Tyr Thr Leu Thr Asn Arg Asp LeuArg His Ala Leu Leu Arg Leu 305 310 315 320 gtc tgc tgc gga cgc cac tcctgc ggc aga gac ccg agt ggc tcc cag 1008 Val Cys Cys Gly Arg His Ser CysGly Arg Asp Pro Ser Gly Ser Gln 325 330 335 cag tcg gcg agc gcg gct gaggct tcc ggg ggc ctg cgc cgc tgc ctg 1056 Gln Ser Ala Ser Ala Ala Glu AlaSer Gly Gly Leu Arg Arg Cys Leu 340 345 350 ccc ccg ggc ctt gat ggg agcttc agc ggc tcg gag cgc tca tcg ccc 1104 Pro Pro Gly Leu Asp Gly Ser PheSer Gly Ser Glu Arg Ser Ser Pro 355 360 365 cag cgc gac ggg ctg gac accagc ggc tcc aca ggc agc ccc ggt gca 1152 Gln Arg Asp Gly Leu Asp Thr SerGly Ser Thr Gly Ser Pro Gly Ala 370 375 380 ccc aca gcc gcc cgg act ctggta tca gaa ccg gct gca gac tga 1197 Pro Thr Ala Ala Arg Thr Leu Val SerGlu Pro Ala Ala Asp 385 390 395 2 398 PRT Homo sapiens 2 Met Glu Ser GlyLeu Leu Arg Pro Ala Pro Val Ser Glu Val Ile Val 1 5 10 15 Leu His TyrAsn Tyr Thr Gly Lys Leu Arg Gly Ala Arg Tyr Gln Pro 20 25 30 Gly Ala GlyLeu Arg Ala Asp Ala Val Val Cys Leu Ala Val Cys Ala 35 40 45 Phe Ile ValLeu Glu Asn Leu Ala Val Leu Leu Val Leu Gly Arg His 50 55 60 Pro Arg PheHis Ala Pro Met Phe Leu Leu Leu Gly Ser Leu Thr Leu 65 70 75 80 Ser AspLeu Leu Ala Gly Ala Ala Tyr Ala Ala Asn Ile Leu Leu Ser 85 90 95 Gly ProLeu Thr Leu Lys Leu Ser Pro Ala Leu Trp Phe Ala Arg Glu 100 105 110 GlyGly Val Phe Val Ala Leu Thr Ala Ser Val Leu Ser Leu Leu Ala 115 120 125Ile Ala Leu Glu Arg Ser Leu Thr Met Ala Arg Arg Gly Pro Ala Pro 130 135140 Val Ser Ser Arg Gly Arg Thr Leu Ala Met Ala Ala Ala Ala Trp Gly 145150 155 160 Val Ser Leu Leu Leu Gly Leu Leu Pro Ala Leu Gly Trp Asn CysLeu 165 170 175 Gly Arg Leu Asp Ala Cys Ser Thr Val Leu Pro Leu Tyr AlaLys Ala 180 185 190 Tyr Val Leu Phe Cys Val Leu Ala Phe Val Gly Ile LeuAla Ala Ile 195 200 205 Cys Ala Leu Tyr Ala Arg Ile Tyr Cys Gln Val ArgAla Asn Ala Arg 210 215 220 Arg Leu Pro Ala Arg Pro Gly Thr Ala Gly ThrThr Ser Thr Arg Ala 225 230 235 240 Arg Arg Lys Pro Arg Ser Leu Ala LeuLeu Arg Thr Leu Ser Val Val 245 250 255 Leu Leu Ala Phe Val Ala Cys TrpGly Pro Leu Phe Leu Leu Leu Leu 260 265 270 Leu Asp Val Ala Cys Pro AlaArg Thr Cys Pro Val Leu Leu Gln Ala 275 280 285 Asp Pro Phe Leu Gly LeuAla Met Ala Asn Ser Leu Leu Asn Pro Ile 290 295 300 Ile Tyr Thr Leu ThrAsn Arg Asp Leu Arg His Ala Leu Leu Arg Leu 305 310 315 320 Val Cys CysGly Arg His Ser Cys Gly Arg Asp Pro Ser Gly Ser Gln 325 330 335 Gln SerAla Ser Ala Ala Glu Ala Ser Gly Gly Leu Arg Arg Cys Leu 340 345 350 ProPro Gly Leu Asp Gly Ser Phe Ser Gly Ser Glu Arg Ser Ser Pro 355 360 365Gln Arg Asp Gly Leu Asp Thr Ser Gly Ser Thr Gly Ser Pro Gly Ala 370 375380 Pro Thr Ala Ala Arg Thr Leu Val Ser Glu Pro Ala Ala Asp 385 390 3953 364 PRT Homo sapiens 3 Met Ala Ala Ile Ser Thr Ser Ile Pro Val Ile SerGln Pro Gln Phe 1 5 10 15 Thr Ala Met Asn Glu Pro Gln Cys Phe Tyr AsnGlu Ser Ile Ala Phe 20 25 30 Phe Tyr Asn Arg Ser Gly Lys His Leu Ala ThrGlu Trp Asn Thr Val 35 40 45 Ser Lys Leu Val Met Gly Leu Gly Ile Thr ValCys Ile Phe Ile Met 50 55 60 Leu Ala Asn Leu Leu Val Met Val Ala Ile TyrVal Asn Arg Arg Phe 65 70 75 80 His Phe Pro Ile Tyr Tyr Leu Met Ala AsnLeu Ala Ala Ala Asp Phe 85 90 95 Phe Ala Gly Leu Ala Tyr Phe Tyr Leu MetPhe Asn Thr Gly Pro Asn 100 105 110 Thr Arg Arg Leu Thr Val Ser Thr TrpLeu Leu Arg Gln Gly Leu Ile 115 120 125 Asp Thr Ser Leu Thr Ala Ser ValAla Asn Leu Leu Ala Ile Ala Ile 130 135 140 Glu Arg His Ile Thr Val PheArg Met Gln Leu His Thr Arg Met Ser 145 150 155 160 Asn Arg Arg Val ValVal Val Ile Val Val Ile Trp Thr Met Ala Ile 165 170 175 Val Met Gly AlaIle Pro Ser Val Gly Trp Asn Cys Ile Cys Asp Ile 180 185 190 Glu Asn CysSer Asn Met Ala Pro Leu Tyr Ser Asp Ser Tyr Leu Val 195 200 205 Phe TrpAla Ile Phe Asn Leu Val Thr Phe Val Val Met Val Val Leu 210 215 220 TyrAla His Ile Phe Gly Tyr Val Arg Gln Arg Thr Met Arg Met Ser 225 230 235240 Arg His Ser Ser Gly Pro Arg Arg Asn Arg Asp Thr Met Met Ser Leu 245250 255 Leu Lys Thr Val Val Ile Val Leu Gly Ala Phe Ile Ile Cys Trp Thr260 265 270 Pro Gly Leu Val Leu Leu Leu Leu Asp Val Cys Cys Pro Gln CysAsp 275 280 285 Val Leu Ala Tyr Glu Lys Phe Phe Leu Leu Leu Ala Glu PheAsn Ser 290 295 300 Ala Met Asn Pro Ile Ile Tyr Ser Tyr Arg Asp Lys GluMet Ser Ala 305 310 315 320 Thr Phe Arg Gln Ile Leu Cys Cys Gln Arg SerGlu Asn Pro Thr Gly 325 330 335 Pro Thr Glu Ser Ser Asp Arg Ser Ala SerSer Leu Asn His Thr Ile 340 345 350 Leu Ala Gly Val His Ser Asn Asp HisSer Val Val 355 360 4 353 PRT Homo sapiens 4 Met Asn Glu Cys His Tyr AspLys His Met Asp Phe Phe Tyr Asn Arg 1 5 10 15 Ser Asn Thr Asp Thr ValAsp Asp Trp Thr Gly Thr Lys Leu Val Ile 20 25 30 Val Leu Cys Val Gly ThrPhe Phe Cys Leu Phe Ile Phe Phe Ser Asn 35 40 45 Ser Leu Val Ile Ala AlaVal Ile Lys Asn Arg Lys Phe His Phe Pro 50 55 60 Phe Tyr Tyr Leu Leu AlaAsn Leu Ala Ala Ala Asp Phe Phe Ala Gly 65 70 75 80 Ile Ala Tyr Val PheLeu Met Phe Asn Thr Gly Pro Val Ser Lys Thr 85 90 95 Leu Thr Val Asn ArgTrp Phe Leu Arg Gln Gly Leu Leu Asp Ser Ser 100 105 110 Leu Thr Ala SerLeu Thr Asn Leu Leu Val Ile Ala Val Glu Arg His 115 120 125 Met Ser IleMet Arg Met Arg Val His Ser Asn Leu Thr Lys Lys Arg 130 135 140 Val ThrLeu Leu Ile Leu Leu Val Trp Ala Ile Ala Ile Phe Met Gly 145 150 155 160Ala Val Pro Thr Leu Gly Trp Asn Cys Leu Cys Asn Ile Ser Ala Cys 165 170175 Ser Ser Leu Ala Pro Ile Tyr Ser Arg Ser Tyr Leu Val Phe Trp Thr 180185 190 Val Ser Asn Leu Met Ala Phe Leu Ile Met Val Val Val Tyr Leu Arg195 200 205 Ile Tyr Val Tyr Val Lys Arg Lys Thr Asn Val Leu Ser Pro HisThr 210 215 220 Ser Gly Ser Ile Ser Arg Arg Arg Thr Pro Met Lys Leu MetLys Thr 225 230 235 240 Val Met Thr Val Leu Gly Ala Phe Val Val Cys TrpThr Pro Gly Leu 245 250 255 Val Val Leu Leu Leu Asp Gly Leu Asn Cys ArgGln Cys Gly Val Gln 260 265 270 His Val Lys Arg Trp Phe Leu Leu Leu AlaLeu Leu Asn Ser Val Val 275 280 285 Asn Pro Ile Ile Tyr Ser Tyr Lys AspGlu Asp Met Tyr Gly Thr Met 290 295 300 Lys Lys Met Ile Cys Cys Phe SerGln Glu Asn Pro Glu Arg Arg Pro 305 310 315 320 Ser Arg Ile Pro Ser ThrVal Leu Ser Arg Ser Asp Thr Gly Ser Gln 325 330 335 Tyr Ile Glu Asp SerIle Ser Gln Gly Ala Val Cys Asn Lys Ser Thr 340 345 350 Ser 5 382 PRTHomo sapiens 5 Met Val Ile Met Gly Gln Cys Tyr Tyr Asn Glu Thr Ile GlyPhe Phe 1 5 10 15 Tyr Asn Asn Ser Gly Lys Glu Leu Ser Ser His Trp ArgPro Lys Asp 20 25 30 Val Val Val Val Ala Leu Gly Leu Thr Val Ser Val LeuVal Leu Leu 35 40 45 Thr Asn Leu Leu Val Ile Ala Ala Ile Ala Ser Asn ArgArg Phe His 50 55 60 Gln Pro Ile Tyr Tyr Leu Leu Gly Asn Leu Ala Ala AlaAsp Leu Phe 65 70 75 80 Ala Gly Val Ala Tyr Leu Phe Leu Met Phe His ThrGly Pro Arg Thr 85 90 95 Ala Arg Leu Ser Leu Glu Gly Trp Phe Leu Arg GlnGly Leu Leu Asp 100 105 110 Thr Ser Leu Thr Ala Ser Val Ala Thr Leu LeuAla Ile Ala Val Glu 115 120 125 Arg His Arg Ser Val Met Ala Val Gln LeuHis Ser Arg Leu Pro Arg 130 135 140 Gly Arg Val Val Met Leu Ile Val GlyVal Trp Val Ala Ala Leu Gly 145 150 155 160 Leu Gly Leu Leu Pro Ala HisSer Trp His Cys Leu Cys Ala Leu Asp 165 170 175 Arg Cys Ser Arg Met AlaPro Leu Leu Ser Arg Ser Tyr Leu Ala Val 180 185 190 Trp Ala Leu Ser SerLeu Leu Val Phe Leu Leu Met Val Ala Val Tyr 195 200 205 Thr Arg Ile PhePhe Tyr Val Arg Arg Arg Val Gln Arg Met Ala Glu 210 215 220 His Val SerCys His Pro Arg Tyr Arg Glu Thr Thr Leu Ser Leu Val 225 230 235 240 LysThr Val Val Ile Ile Leu Gly Ala Phe Val Val Cys Trp Thr Pro 245 250 255Gly Gln Val Val Leu Leu Leu Asp Gly Leu Gly Cys Glu Ser Cys Asn 260 265270 Val Leu Ala Val Glu Lys Tyr Phe Leu Leu Leu Ala Glu Ala Asn Ser 275280 285 Leu Val Asn Ala Ala Val Tyr Ser Cys Arg Asp Ala Glu Met Arg Arg290 295 300 Thr Phe Arg Arg Leu Leu Cys Cys Ala Cys Leu Arg Gln Ser ThrArg 305 310 315 320 Glu Ser Val His Tyr Thr Ser Ser Ala Gln Gly Gly AlaSer Thr Arg 325 330 335 Ile Met Leu Pro Glu Asn Gly His Pro Leu Met ThrPro Pro Phe Ser 340 345 350 Tyr Leu Glu Leu Gln Arg Tyr Ala Ala Ser AsnLys Ser Thr Ala Pro 355 360 365 Asp Asp Leu Trp Val Leu Leu Ala Gln ProAsn Gln Gln Asp 370 375 380 6 381 PRT Homo sapiens 6 Met Gly Pro Thr SerVal Pro Leu Val Lys Ala His Arg Ser Ser Val 1 5 10 15 Ser Asp Tyr ValAsn Tyr Asp Ile Ile Val Arg His Tyr Asn Tyr Thr 20 25 30 Gly Lys Leu AsnIle Ser Ala Asp Lys Glu Asn Ser Ile Lys Leu Thr 35 40 45 Ser Val Val PheIle Leu Ile Cys Cys Phe Ile Ile Leu Glu Asn Ile 50 55 60 Phe Val Leu LeuThr Ile Trp Lys Thr Lys Lys Phe His Arg Pro Met 65 70 75 80 Tyr Tyr PheIle Gly Asn Leu Ala Leu Ser Asp Leu Leu Ala Gly Val 85 90 95 Ala Tyr ThrAla Asn Leu Leu Leu Ser Gly Ala Thr Thr Tyr Lys Leu 100 105 110 Thr ProAla Gln Trp Phe Leu Arg Glu Gly Ser Met Phe Val Ala Leu 115 120 125 SerAla Ser Val Phe Ser Leu Leu Ala Ile Ala Ile Glu Arg Tyr Ile 130 135 140Thr Met Leu Lys Met Lys Leu His Asn Gly Ser Asn Asn Phe Arg Leu 145 150155 160 Phe Leu Leu Ile Ser Ala Cys Trp Val Ile Ser Leu Ile Leu Gly Gly165 170 175 Leu Pro Ile Met Gly Trp Asn Cys Ile Ser Ala Leu Ser Ser CysSer 180 185 190 Thr Val Leu Pro Leu Tyr His Lys His Tyr Ile Leu Phe CysThr Thr 195 200 205 Val Phe Thr Leu Leu Leu Leu Ser Ile Val Ile Leu TyrCys Arg Ile 210 215 220 Tyr Ser Leu Val Arg Thr Arg Ser Arg Arg Leu ThrPhe Arg Lys Asn 225 230 235 240 Ile Ser Lys Ala Ser Arg Ser Ser Glu AsnVal Ala Leu Leu Lys Thr 245 250 255 Val Ile Ile Val Leu Ser Val Phe IleAla Cys Trp Ala Pro Leu Phe 260 265 270 Ile Leu Leu Leu Leu Asp Val GlyCys Lys Val Lys Thr Cys Asp Ile 275 280 285 Leu Phe Arg Ala Glu Tyr PheLeu Val Leu Ala Val Leu Asn Ser Gly 290 295 300 Thr Asn Pro Ile Ile TyrThr Leu Thr Asn Lys Glu Met Arg Arg Ala 305 310 315 320 Phe Ile Arg IleMet Ser Cys Cys Lys Cys Pro Ser Gly Asp Ser Ala 325 330 335 Gly Lys PheLys Arg Pro Ile Ile Ala Gly Met Glu Phe Ser Arg Ser 340 345 350 Lys SerAsp Asn Ser Ser His Pro Gln Lys Asp Glu Gly Asp Asn Pro 355 360 365 GluThr Ile Met Ser Ser Gly Asn Val Asn Ser Ser Ser 370 375 380 7 378 PRTHomo sapiens 7 Met Ala Thr Ala Leu Pro Pro Arg Leu Gln Pro Val Arg GlyAsn Glu 1 5 10 15 Thr Leu Arg Glu His Tyr Gln Tyr Val Gly Lys Leu AlaGly Arg Leu 20 25 30 Lys Glu Ala Ser Glu Gly Ser Thr Leu Thr Thr Val LeuPhe Leu Val 35 40 45 Ile Cys Ser Phe Ile Val Leu Glu Asn Leu Met Val LeuIle Ala Ile 50 55 60 Trp Lys Asn Asn Lys Phe His Asn Arg Met Tyr Phe PheIle Gly Asn 65 70 75 80 Leu Ala Leu Cys Asp Leu Leu Ala Gly Ile Ala TyrLys Val Asn Ile 85 90 95 Leu Met Ser Gly Lys Lys Thr Phe Ser Leu Ser ProThr Val Trp Phe 100 105 110 Leu Arg Glu Gly Ser Met Phe Val Ala Leu GlyAla Ser Thr Cys Ser 115 120 125 Leu Leu Ala Ile Ala Ile Glu Arg His LeuThr Met Ile Lys Met Arg 130 135 140 Pro Tyr Asp Ala Asn Lys Arg His ArgVal Phe Leu Leu Ile Gly Met 145 150 155 160 Cys Trp Leu Ile Ala Phe ThrLeu Gly Ala Leu Pro Ile Leu Gly Trp 165 170 175 Asn Cys Leu His Asn LeuPro Asp Cys Ser Thr Ile Leu Pro Leu Tyr 180 185 190 Ser Lys Lys Tyr IleAla Phe Cys Ile Ser Ile Phe Thr Ala Ile Leu 195 200 205 Val Thr Ile ValIle Leu Tyr Ala Arg Ile Tyr Phe Leu Val Lys Ser 210 215 220 Ser Ser ArgLys Val Ala Asn His Asn Asn Ser Glu Arg Ser Met Ala 225 230 235 240 LeuLeu Arg Thr Val Val Ile Val Val Ser Val Phe Ile Ala Cys Trp 245 250 255Ser Pro Leu Phe Ile Leu Phe Leu Ile Asp Val Ala Cys Arg Val Gln 260 265270 Ala Cys Pro Ile Leu Phe Lys Ala Gln Trp Phe Ile Val Leu Ala Val 275280 285 Leu Asn Ser Ala Met Asn Pro Val Ile Tyr Thr Leu Ala Ser Lys Glu290 295 300 Met Arg Arg Ala Phe Phe Arg Leu Val Cys Asn Cys Leu Val ArgGly 305 310 315 320 Arg Gly Ala Arg Ala Ser Pro Ile Gln Pro Ala Leu AspPro Ser Arg 325 330 335 Ser Lys Ser Ser Ser Ser Asn Asn Ser Ser His SerPro Lys Val Lys 340 345 350 Glu Asp Leu Pro His Thr Asp Pro Ser Ser CysIle Met Asp Lys Asn 355 360 365 Ala Ala Leu Gln Asn Gly Ile Phe Cys Asn370 375 8 353 PRT Homo sapiens 8 Met Gly Ser Leu Tyr Ser Glu Tyr Leu AsnPro Asn Lys Val Gln Glu 1 5 10 15 His Tyr Asn Tyr Thr Lys Glu Thr LeuGlu Thr Gln Glu Thr Thr Ser 20 25 30 Arg Gln Val Ala Ser Ala Phe Ile ValIle Leu Cys Cys Ala Ile Val 35 40 45 Val Glu Asn Leu Leu Val Leu Ile AlaVal Ala Arg Asn Ser Lys Phe 50 55 60 His Ser Ala Met Tyr Leu Phe Leu GlyAsn Leu Ala Ala Ser Asp Leu 65 70 75 80 Leu Ala Gly Val Ala Phe Val AlaAsn Thr Leu Leu Ser Gly Ser Val 85 90 95 Thr Leu Arg Leu Thr Pro Val GlnTrp Phe Ala Arg Glu Gly Ser Ala 100 105 110 Ser Ile Thr Leu Ser Ala SerVal Phe Ser Leu Leu Ala Ile Ala Ile 115 120 125 Glu Arg His Val Ala IleAla Lys Val Lys Leu Tyr Gly Ser Asp Lys 130 135 140 Ser Cys Arg Met LeuLeu Leu Ile Gly Ala Ser Trp Leu Ile Ser Leu 145 150 155 160 Val Leu GlyGly Leu Pro Ile Leu Gly Trp Asn Cys Leu Gly His Leu 165 170 175 Glu AlaCys Ser Thr Val Leu Pro Leu Tyr Ala Lys His Tyr Val Leu 180 185 190 CysVal Val Thr Ile Phe Ser Ile Ile Leu Leu Ala Ile Val Ala Leu 195 200 205Tyr Val Arg Ile Tyr Cys Val Val Arg Ser Ser His Ala Asp Met Ala 210 215220 Ala Pro Gln Thr Leu Ala Leu Leu Lys Thr Val Thr Ile Val Leu Gly 225230 235 240 Val Phe Ile Val Cys Trp Leu Pro Ala Phe Ser Ile Leu Leu LeuAsp 245 250 255 Tyr Ala Cys Pro Val His Ser Cys Pro Ile Leu Tyr Lys AlaHis Tyr 260 265 270 Phe Phe Ala Val Ser Thr Leu Asn Ser Leu Leu Asn ProVal Ile Tyr 275 280 285 Thr Trp Arg Ser Arg Asp Leu Arg Arg Glu Val LeuArg Pro Leu Gln 290 295 300 Cys Trp Arg Pro Gly Val Gly Val Gln Gly ArgArg Arg Val Gly Thr 305 310 315 320 Pro Gly His His Leu Leu Pro Leu ArgSer Ser Ser Ser Leu Glu Arg 325 330 335 Gly Met His Met Pro Thr Ser ProThr Phe Leu Glu Gly Asn Thr Val 340 345 350 Val 9 384 PRT Homo sapiens 9Met Asn Ala Thr Gly Thr Pro Val Ala Pro Glu Ser Cys Gln Gln Leu 1 5 1015 Ala Ala Gly Gly His Ser Arg Leu Ile Val Leu His Tyr Asn His Ser 20 2530 Gly Arg Leu Ala Gly Arg Gly Gly Pro Glu Asp Gly Gly Leu Gly Ala 35 4045 Leu Arg Gly Leu Ser Val Ala Ala Ser Cys Leu Val Val Leu Glu Asn 50 5560 Leu Leu Val Leu Ala Ala Ile Thr Ser His Met Arg Ser Arg Arg Trp 65 7075 80 Val Tyr Tyr Cys Leu Val Asn Ile Thr Leu Ser Asp Leu Leu Thr Gly 8590 95 Ala Ala Tyr Leu Ala Asn Val Leu Leu Ser Gly Ala Arg Thr Phe Arg100 105 110 Leu Ala Pro Ala Gln Trp Phe Leu Arg Glu Gly Leu Leu Phe ThrAla 115 120 125 Leu Ala Ala Ser Thr Phe Ser Leu Leu Phe Thr Ala Gly GluArg Phe 130 135 140 Ala Thr Met Val Arg Pro Val Ala Glu Ser Gly Ala ThrLys Thr Ser 145 150 155 160 Arg Val Tyr Gly Phe Ile Gly Leu Cys Trp LeuLeu Ala Ala Leu Leu 165 170 175 Gly Met Leu Pro Leu Leu Gly Trp Asn CysLeu Cys Ala Phe Asp Arg 180 185 190 Cys Ser Ser Leu Leu Pro Leu Tyr SerLys Arg Tyr Ile Leu Phe Cys 195 200 205 Leu Val Ile Phe Ala Gly Val LeuAla Thr Ile Met Gly Leu Tyr Gly 210 215 220 Ala Ile Phe Arg Leu Val GlnAla Ser Gly Gln Lys Ala Pro Arg Pro 225 230 235 240 Ala Ala Arg Arg LysAla Arg Arg Leu Leu Lys Thr Val Leu Met Ile 245 250 255 Leu Leu Ala PheLeu Val Cys Trp Gly Pro Leu Phe Gly Leu Leu Leu 260 265 270 Ala Asp ValPhe Gly Ser Asn Leu Trp Ala Gln Glu Tyr Leu Arg Gly 275 280 285 Met AspTrp Ile Leu Ala Leu Ala Val Leu Asn Ser Ala Val Asn Pro 290 295 300 IleIle Tyr Ser Phe Arg Ser Arg Glu Val Cys Arg Ala Val Leu Ser 305 310 315320 Phe Leu Cys Cys Gly Cys Leu Arg Leu Gly Met Arg Gly Pro Gly Asp 325330 335 Cys Leu Ala Arg Ala Val Glu Ala His Ser Gly Ala Ser Thr Thr Asp340 345 350 Ser Ser Leu Arg Pro Arg Asp Ser Phe Arg Gly Ser Arg Ser LeuSer 355 360 365 Phe Arg Met Arg Glu Pro Leu Ser Ser Ile Ser Ser Val ArgSer Ile 370 375 380

What is claimed is:
 1. An isolated polynucleotide comprising apolynucleotide selected from the group consisting of: (a) apolynucleotide encoding the polypeptide consisting of the amino acidsequence of SEQ ID NO:2; (b) a polynucleotide consisting of SEQ ID NO:1;(c) a polynucleotide having at least about 90% sequence identity to thepolynucleotide of (a) or (b).
 2. The isolated polynucleotide of claim 1,which comprises a polynucleotide having at least about 90% sequenceidentity to SEQ ID NO:
 1. 3. The isolated polynucleotide of claim 1,which comprises a polynucleotide having at least about 90% sequenceidentity to a polynucleotide encoding the polypeptide as set forth inSEQ ID NO:2.
 4. The isolated polynucleotide of claim 1, which comprisesa polynucleotide having at least about 95% sequence identity to apolynucleotide encoding SEQ ID NO:2.
 5. The isolated polynucleotide ofclaim 1, which comprises a polynucleotide encoding SEQ ID NO:2.
 6. Thepolynucleotide of claim 1, wherein said polynucleotide comprises SEQ IDNO:1.
 7. The polynucleotide of claim 1, wherein said polynucleotidesequence encodes the polypeptide of SEQ ID NO:2.
 8. The polynucleotideof claim 1, which is a DNA or RNA.
 9. A fragment of the polynucleotideof SEQ ID NO:1.
 10. An expression vector comprising the isolatedpolynucleotide of claim
 1. 11. A host cell comprising the expressionvector of claim
 10. 12. The host cell of claim 10, which is a mammaliancell.
 13. The host cell of claim 10, wherein the mammalian cell is a CHOcell.
 14. The host cell of claim 10, which is a eukaryotic cell.
 15. Anantibody that selectively binds a polypeptide comprising the amino acidsequence of SEQ ID NO:2 or a fragment thereof.
 16. A process forproducing the polypeptide comprising SEQ ID NO: 2 comprising: culturinga host cell of claim 11 under conditions sufficient for the productionof said polypeptide and recovering the polypeptide from the culture. 17.A process for producing cells capable of expressing a polypeptidecomprising genetically transfecting or transforming cells with thevector of claim
 10. 18. A process for producing a human EDG8 polypeptideor a fragment thereof comprising: culturing a host cell of claim 11under conditions sufficient for the production of said polypeptide andrecovering the polypeptide from the culture.
 19. A polynucleotide whichis a complement of a polynucleotide of claim
 1. 20. A process fordiagnosing a disease or a susceptibility to a disease related toexpression or activity of human EDG8 polypeptide comprising: determiningthe presence or absence of mutation in the nucleotide sequence encodingsaid human EDG8 polypeptide in the genome of said subject; and/oranalyzing for the presence or amount of the human EDG8 polypeptideexpression in a sample derived from said subject.
 21. A method foridentifying compounds which bind to human EDG8 polypeptide comprising:a) contacting a cell as claimed in claim 18 or a part thereof with acandidate compound; and b) assessing the ability of said candidatecompound to bind to said cells.
 22. The method as claimed in claim 21which further includes determining whether the candidate compoundeffects a signal generated by activation of the human EDG8 polypeptideat the surface of the cell, wherein a candidate compound which effectsproduction of said signal is identified as an agonist.
 23. The method asclaimed in claim 21 which further includes determining whether thecandidate compound effects a signal generated by activation of the humanEDG8 polypeptide at the surface of the cell, wherein a candidatecompound which effects production of said signal is identified as anantagonist.
 24. An agonist identified by the method of claim
 22. 25. Anantagonist identified by the method of claim
 23. 26. The method of claim21 which further includes contacting said cell with a known agonist forsaid human EDG8 polypeptide; and determining whether the signalgenerated by said agonist is diminished in the presence of saidcandidate compound, wherein a candidate compound which effects adiminution in said signal is identified as an antagonist for said humanEDG8 polypeptide.
 27. A method as claimed in claim 26, wherein the knownagonist is S1P, LPA and/or dHS1P.
 28. An antagonist identified by themethod of claim
 26. 29. A method of preparing a pharmaceuticalcomposition comprising: a) identifying a compound which is an agonist oran antagonist of human EDG8, b) preparing the compound, and c)optionally mixing the compound with suitable additives.
 30. Apharmaceutical composition prepared by a method of claim
 29. 31. Apharmaceutical composition comprising human EDG8 polypeptide or afragment thereof wherein said fragment has human EDG8 biologicalactivity.
 32. A pharmaceutical composition containing a polynucleotideencoding for human EDG8 or a fragment thereof encoding for a peptidewith human EDG8 biological activity.